U.S. patent number 8,343,536 [Application Number 12/019,477] was granted by the patent office on 2013-01-01 for biofilm-inhibiting medical products.
This patent grant is currently assigned to Cook Biotech Incorporated, Cook Medical Technologies LLC. Invention is credited to Brian L. Bates, Michael C. Hiles, Chad E. Johnson.
United States Patent |
8,343,536 |
Bates , et al. |
January 1, 2013 |
**Please see images for:
( Certificate of Correction ) ** |
Biofilm-inhibiting medical products
Abstract
A biofilm-inhibiting medical product includes a carrier formed
from a natural, bioremodelable material, whereby the carrier
includes a biocidal bismuth thiol agent and/or one or more other
biofilm-inhibiting or wound healing agents. A method for using the
biofilm-inhibiting medical product to treat a wound or tissue
defect in a patient's body is described.
Inventors: |
Bates; Brian L. (Bloomington,
IN), Hiles; Michael C. (Lafayette, IN), Johnson; Chad
E. (West Lafayette, IN) |
Assignee: |
Cook Biotech Incorporated (West
Layfayette, IN)
Cook Medical Technologies LLC (Bloomington, IN)
|
Family
ID: |
39668277 |
Appl.
No.: |
12/019,477 |
Filed: |
January 24, 2008 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20080181950 A1 |
Jul 31, 2008 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
60897114 |
Jan 24, 2007 |
|
|
|
|
Current U.S.
Class: |
424/445; 435/325;
424/520 |
Current CPC
Class: |
A61P
17/02 (20180101); A61L 15/40 (20130101); A61L
15/44 (20130101); A61L 2300/404 (20130101); A61L
2300/102 (20130101) |
Current International
Class: |
A61L
15/00 (20060101); A61K 35/12 (20060101); C12N
15/00 (20060101) |
Field of
Search: |
;424/445,520
;435/325 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
1526778 |
|
Sep 1978 |
|
GB |
|
WO 98/22158 |
|
May 1998 |
|
WO |
|
WO 98/58075 |
|
Dec 1998 |
|
WO |
|
WO 99/21568 |
|
May 1999 |
|
WO |
|
WO 99/39707 |
|
Aug 1999 |
|
WO |
|
WO 00/07638 |
|
Feb 2000 |
|
WO |
|
WO 00/33895 |
|
Jun 2000 |
|
WO |
|
WO 00/71139 |
|
Nov 2000 |
|
WO |
|
WO 03/002165 |
|
Jan 2003 |
|
WO |
|
WO 03/011821 |
|
Feb 2003 |
|
WO |
|
WO 03/035125 |
|
May 2003 |
|
WO |
|
WO 03/045294 |
|
Jun 2003 |
|
WO |
|
WO 03/066119 |
|
Aug 2003 |
|
WO |
|
WO 2004/103071 |
|
Dec 2004 |
|
WO |
|
WO 2005/018701 |
|
Mar 2005 |
|
WO |
|
WO 2005/020847 |
|
Mar 2005 |
|
WO |
|
WO 2005/030186 |
|
Apr 2005 |
|
WO |
|
WO 2005/055723 |
|
Jun 2005 |
|
WO |
|
WO 2005/087135 |
|
Sep 2005 |
|
WO |
|
WO 2005/094579 |
|
Oct 2005 |
|
WO |
|
WO 2005/097219 |
|
Oct 2005 |
|
WO |
|
WO 2005/107455 |
|
Nov 2005 |
|
WO |
|
WO 2006/031554 |
|
Mar 2006 |
|
WO |
|
WO 2006/044512 |
|
Apr 2006 |
|
WO |
|
WO 2006/045041 |
|
Apr 2006 |
|
WO |
|
WO 2006/121887 |
|
Nov 2006 |
|
WO |
|
WO 2008/067085 |
|
Jun 2008 |
|
WO |
|
Other References
Zhang et al., Inhibitionof Bacterial Adherance on the Surface of
Stents and Bacterial Grouwth in Bile by Bismuth Dimercaprol, Jun.
2005, Digestive diseases and Sciences, vol. 50, No. 6, pp.
1046-1051. cited by examiner .
Domenico, Philip et al., BisEDT and RIP act in synergy to prevent
graft ingections by resistant staphylococci, 2004, Peptides, vol.
25, pp. 2047-2053. cited by examiner .
Bradley, M. et al., Systematic reviews of wound care management:
(2) Dressings and topical agents used in the healing of chronic
wounds, Health Technology Assessment, vol. 3, No. 17 (Pt. 2),
(1999). cited by other .
Bradley, M. et al., The debridement of chronic wounds: a systematic
review, Health Technology Assessment, vol. 3, No. 17 (Pt. 1),
(1999). cited by other .
Bruns and Worthington (2000) Am. Fam. Physician 61 :1383-1388.
cited by other .
Carmeliet, P., Mechanisms of angiogenesis and arteriogenesis, Nat
Med 6 (2000), No. 4, 389-395. cited by other .
Cullum, N. et al., Systematic reviews of wound care management: (5)
beds; (6) compres-sion; (7) laser therapy and electromagnetic
therapy, Health Technology Assessment, vol. 5, No. 9, (2001). cited
by other .
Davey, M. E. et al., Microbial Biofilms: from Ecology to Molecular
Genetics, Microbiology and Molecular Biology Reviews, vol. 64, No.
4, p. 847-867, (Dec. 2000). cited by other .
Domenico, Philip et al., Activities of Bismuth Thiols against
Staphylococci and Staphylococcal Biofilms, Antimicrobial Agents and
Chemotherapy, vol. 45, No. 5, p. 1417-1421 (May 2001). cited by
other .
Domenico, Philip et al., Enhancement of Bismuth Antibacterial
Activity with Lipophilic Thiol Chelators, Antimicrobial Agents and
Chemotherapy, vol. 41, No. 8, p. 1697-1703 (Aug. 1997). cited by
other .
Domenico, Philip et al., Surface Antigen Exposure by Bismuth
Dimercaprol Suppression of Klebsiella pneumonia Capsular
Polysaccharide, Infection and Immunity, vol. 67, No. 2, p. 664-669
(Feb. 1999). cited by other .
Donlan, Rodney M., et al., Biofilms: Survival Mechanisms of
Clinically Relevant Microorganisms, Clinical Microbiology Reviews,
vol. 15, No. 2, p. 167-193 (Apr. 2002). cited by other .
Drosou, Anna, MD, et al., Feature: Antiseptics on Wounds: An Area
of Controversy (Part One), Wounds vol. 15, Issue 5, p. 149-166 (May
2003). cited by other .
Drosou, Anna, MD, et al., Feature: Antiseptics on Wounds: An Area
of Controversy (Part Two), Wounds, vol. 15, Issue 5, p. 149-166
(May 2003). cited by other .
Gilbert, Peter et al., Potential Impact of Increased Use of
Biocides in Consumer Products on Prevalence of Antibiotic
Resistance, Clinic Microbiology Reviews, vol. 16, No. 2, p. 189-208
(Apr. 2003). cited by other .
Harding, K.G. et al., Healing chronic wounds, BMJ vol. 324, p.
160-3, (Jan. 19, 2002). cited by other .
Heeschen, C. et al., Nat Med vol. 7, No. 7, pp. 833-839, 2001.
cited by other .
Huynh, Nature Biotechnology, vol. 17, p. 1083-1086 (Nov. 1999).
cited by other .
International Search Report Jul. 28, 2009. cited by other .
Johansen, Charlotte et al., Enzymatic Removal and Disinfection of
Bacterial Biofilms, Applied and Environmental Microbiology, vol.
63, No. 9, p. 3724-3728 (Sep. 1997). cited by other .
Johnson, C. et al.,Matrix Metalloproteinase-9 Is Required fro
Adequate Angiogenic Revascularization of Ischemic Tissues:
Potential Role in Capillary Branching, Circ Res., vol. 94, No. 2,
pp. 262-268, 2004. cited by other .
Jugdutt, Bodh I., Ventricular Remodeling After Infarction and the
Extracellular Collagen Matrix: When Is Enough Enough?, Circulation,
108; p. 1395-1403, (2003). cited by other .
Lobmann, Ralf, MD, et al., Proteases and the Diabetic Foot
Syndrome: Mechanisms and Therapeutic Implications, Diabetes Care,
vol. 28, No. 2 (Feb. 2005). cited by other .
MedPro Month (1999) 9:261-262. cited by other .
MedPro Month (2000) 10:86-91. cited by other .
Mertz, Patricia M., Cutaneous Biofilms: Friend or Foe?, Wounds
15(5): p. 129-132, (2003). cited by other .
Monami, M. et al., Use of an Oxidized Regenerated Cellulose and
Collagen Composite for Healing of Chronic Diabetic Foot Ulcers,
Diabetes Care, vol. 25, No. 10, (Oct. 2002). cited by other .
Munro, Neil et al., Infections in the diabetic foot, A practical
management guide to foot care, The British Journal of Diabetes and
Vascular Disease, vol. 3, Issue 2, p. 132-6, (Mar./Apr. 2003).
cited by other .
O'Gara, James et al., Staphylococcus epidermidis biofilms:
importance and implications, J. Med. Microbiol., vol. 50, p.
582-587 (2001). cited by other .
O'Meara, S. et al., Systematic reviews of wound care management:
(3) antimicrobial agents for chronic wounds; (4) diabetic foot
ulceration, Health Technology Assessment, vol. 4, No. 21 (2000).
cited by other .
Ryder, Marcia A, Catheter-Related Infections: It's All About
Biofilm, Topics in Advanced Practice Nursing 5(3) (Posted Aug. 18,
2005). cited by other .
Scardillo, J. Managing tubes and drains: Considerations for
infection control, Infection Control Resource, vol. 2, No. 1, p.
2-3, 6-7, (2003). cited by other .
Steffensen, Bjorn et al., Proteolytic Events of
Wound-Healing--Coordinated Interactions Among Matrix
Metalloproteinases (MMPs), Integrins, and Extracellular Matrix
Molecules, Crit Rev Oral Biol Med, 12(5), p. 373-398 (2001). cited
by other .
Tapiainen, Terhi et al., Ultrastructure of Streptococcus pneumonia
after exposure to xylitol, Journal of Antimicrobial Chemotherapy,
54, p. 225-228 (2004). cited by other .
Tomaselli, Nancy, Prevention and treatment of surgical-site
infections, Infection Control Resource, vol. 2, No. 1, p. 1, 4-6,
(2003). cited by other .
Veloira, W. G. et al., In vitro activity and synergy aof bismuth
thiols and tobramycin against Burkholderia cepacia complex, J.
Antimicrobial chemotherapy, 52, p. 915-919, (2003). cited by other
.
Wu, Chieh-Liang et al., Subinhibitory Bismuth-Thiols Reduce
Virulence of Pseudomonas aeruginosa, Am. J. Respir. Cell. Mol.
Biol., vol. 26, p. 731-738 (2002). cited by other .
Zhang et al.; Digestive Diseases and Sciences, vol. 50, No. 6 (Jun.
1, 2006) pp. 1046-1051. cited by other.
|
Primary Examiner: Roberts; Lezah
Assistant Examiner: Holloman; Nannette
Attorney, Agent or Firm: Brinks Hofer Gilson & Lione
Parent Case Text
This application claims the benefit of priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application No. 60/897,114, filed
Jan. 24, 2007, which is hereby incorporated by reference in its
entirety.
Claims
The invention claimed is:
1. A medical product comprising: a bismuth thiol, and a
bioremodelable extracellular matrix material comprising a fluidized
submucosal tissue having viscosity of about 2 to about 300,000 cps
at 25.degree. C.; and a biocompatible substrate layer adhered to
the bioremodelable extracellular matrix material.
2. The medical product of claim 1, wherein the bismuth thiol is
selected from the group consisting of bismuth-1,2-ethanedithiol,
bismuth-2-mercaptoethanol, bismuth-3,4-dimercaptotoluene,
bismuth-pyrithione, bismuth-2,3-dimercaptopropanol,
bismuth-1,3-propanedithiol, bismuth-dithiothreitol,
bismuth-3-mercapto-2-butanol, and mixtures thereof.
3. The medical product of claim 1, wherein the bismuth thiol is a
bismuth dithiol.
4. The medical product of claim 1, wherein the bismuth dithiol is
bismuth-1,2-ethanedithiol.
5. The medical product of claim 1, wherein the bioremodelable
extracellular matrix material retains at least one growth factor
from a source tissue.
6. The medical product of claim 5, wherein the bioremodelable
extracellular matrix material is in the form of a gel.
7. The of claim 6, wherein the gel is formed from collagenous
submucosal tissue material.
8. The medical product of claim 1, further comprising at least one
biofilm-inhibiting agent selected from the group consisting of
lactoferrin, xylitol, quorum sensing inhibitor, biocidal agent,
antibiotic, surfactant, and mixtures thereof.
9. The medical product of claim 1; where the medical is a hernia
repair device.
10. The medical product of claim 9, wherein the bismuth thiol is
selected from the group consisting of bismuth-1,2-ethanedithiol,
bismuth-2-mercaptoethanol, bismuth-3,4-dimercaptotoluene,
bismuth-pyrithione, bismuth-2,3-dimercaptopropanol,
bismuth-1,3-propanedithiol, bismuth-dithiothreitol,
bismuth-3-mercapto-2-butanol, and mixtures thereof.
11. The medical product of claim 9, wherein the bismuth thiol is a
bismuth dithiol.
12. The medical product of claim 11, wherein the bismuth dithiol is
bismuth-1,2-ethanedithiol.
13. The medical product of claim 9, further comprising at least one
biofilm-inhibiting agent selected from the group consisting of
lactoferrin, xylitol, quorum sensing inhibitor, biocidal agent,
antibiotic, surfactant, and mixtures thereof.
14. The medical product of claim 1; wherein the medical product is
a bioremodelable wound dressing.
15. The medical product of claim 1, wherein the bismuth thiol is
co-mixed with the bioremodelable extracellular matrix material.
16. The medical product of claim 1, wherein the bioremodelable
extracellular matrix material carries bismuth thiol.
Description
TECHNICAL FIELD
This invention is directed to biofilm-inhibiting medical products
(e.g., wound dressings) including a carrier formed from a natural,
bioremodelable material, the carrier including a biocidal bismuth
thiol agent and/or one or more other biofilm-inhibiting or wound
healing agents. This invention is also directed to methods of using
the medical product for treating a tissue defect in a patient's
body.
BACKGROUND
A biofilm is a community of sessile, stably attached
microorganisms, especially bacteria, embedded in a hydrated matrix
of extracellular polymeric substances exhibiting growth properties
that are distinguished from those of planktonic, free-living
microorganisms. Biofilms represent a target of new compositions for
inhibiting, reducing, preventing, and removing microbial
infections, and are believed to be partly responsible for
increasing the rates of antibiotic resistance. It is thought that
upwards of 60% of all nosocomial (hospital-derived) infections are
due to biofilms, whose role in contaminating medical implants is
now well established. Central venous catheters (CVCs) pose the
greatest risk of device-related infections with infection rates of
3 to 5% and account for the most serious and costly
healthcare-associated infections (See for example, Donlan and
Costerton, Clin. Microbiol. Rev., Vol. 15, No. 2, pp. 167-193,
2002; Davey and O'Toole, Microbiol. Mol. Biol. Rev., Vol. 64, No.
4, pp. 847-867, 2000).
One approach to managing biofilm infections is to identify the
microorganism(s) in the biofilm and to find antibiotic or biocidal
agents capable of killing the microorganisms. A major limitation of
this approach is that models for testing the efficacy of these
agents to not sufficiently represent a biofilm environment. Biofilm
bacteria can be up to 1,000-fold more resistant to antibiotic
treatment than the same organism grown planktonically. Biofilm
bacteria are also more resistant to biocides, such as peroxide,
bleach, acids, and other biocidal agents.
In spite of the dramatic differences in susceptibility to
antimicrobial agents between planktonic and sessile, biofilm
microorganisms, current approaches for targeting biofilm
microorganisms are insufficient in addressing this discrepancy.
Antimicrobial efficacy testing often employs standard broth
microdilution methods reflecting antibiotic efficacy in planktonic,
rather than biofilm systems. Accordingly, broad numbers of
prospective antibiotic- and biocidal agents have been identified
without any expectation of success in the more "real" biofilm
world.
The mechanisms by which resistance to antibiotic or biocidal agents
is achieved remain subject to speculation. It is now known,
however, that the structural organization of biofilms hinders the
ability of antibiotics or biocides to access biofilm bacteria and
can protect bacteria from a host's immune system. Clinical biofilm
infections are marked by recurring symptoms after repeated antibody
treatments. Such treatments typically eliminate planktonic
microorganisms, but allow sessile, biofilm microorganisms to
propagate and disseminate upon termination of antibiotic
therapy.
In recent years, biofilm-based infections attributed to medical
devices, such as catheters, prosthetic heart valves, contact
lenses, and intrauterine devices have received increased attention.
Despite circumstantial evidence suggesting biofilms to be a major
culprit responsible for chronic wounds, their role in chronic
wounds remains poorly understood.
Chronic wounds are open wounds that are recalcitrant to healing.
Chronic wounds are painful, diminish the quality of life, impair
mobility, and frequently lead to amputations. And they present an
enormous financial toll worldwide. In 2004, diabetic foot ulcers
accounted for $10 billion in direct costs (about 4% of the total
personal health spending) and another $5 billion in indirect costs
(disability, nursing homes, etc.). In the U.S., chronic wounds
affect roughly 3 million people and are increasing at exponential
rates, doubling every 4-5 years.
Chronic wounds have a number of barriers which limit healing. Many
of these barriers have been extensively studied, including poor
perfusion, white cell dysfunction, poor nutrition, and repetitive
pressure among others. Although wound beds are known to be
populated by biofilms, their role in abrogating or delaying wound
healing has not yet been experimentally established. In addition,
there are questions regarding the extent to which the cellular
regeneration processes accompanying the healing may inadvertently
provide nutritional support for sustaining biofilm viability.
In light of the foregoing, including the ongoing problems with
conventional wound healing treatments, there is a need for improved
compositions and methods for treating chronic wound bed biofilms
and for adequately balancing the tissue regeneration demands
necessary for achieving full and timely healing wounds,
particularly chronic wounds.
SUMMARY
In one aspect, a wound dressing or other medical product includes a
carrier formed from a natural, bioremodelable material, whereby the
carrier further includes, and is formulated to deliver, a biocidal
bismuth thiol agent and/or other biofilm-inhibiting or wound
healing agents. In a preferred embodiment the carrier is formed
from extracellular matrix (ECM) material and the bismuth thiol is
bismuth-1,2-ethanedithiol.
In another aspect, a method of making a wound dressing or other
medical product includes providing a carrier in the form of a
natural, bioremodelable material and incorporating a bismuth thiol
into or onto at least a portion of the carrier.
In a further aspect, a method of treating a wound or other bodily
defect includes contacting a wound or other site in need of
treatment with a wound dressing or medical product of the present
invention. In one preferred embodiment, the method includes
treatment of a chronic wound with a wound dressing containing a
bismuth dithiol and/or one or more other biofilm-inhibiting or
wound healing agents.
In another aspect, a hernia repair device and method are provided.
The hernia repair device includes a sheet of a natural,
bioremodelable material. The device further includes, and is
formulated to deliver, a biocidal bismuth thiol agent. In a
preferred embodiment the device comprises at least one
extracellular matrix (ECM) material, and the bismuth thiol is
bismuth-1,2-ethanedithiol.
When applied to a wound or other tissue defect, the dressing
enhances treatment by preventing or reducing biofilm formation or
development. Whereas biofilm-inhibiting agents serve to prevent,
reduce, and/or eliminate biofilm microorganisms in a wound, the
bioremodelable matrix provides a material source to promote wound
healing and tissue rebuilding. In particular, the bioremodelable
ECM materials provide a rapidly vascularized matrix to promote the
remodeling and regrowth of endogenous tissue, which ultimately
replaces the exogenously provided ECM-based material.
In the present invention, it is believed that the use of bismuth
thiols in conjunction with bioremodelable materials significantly
improves management of chronic wounds and other tissue defects.
Moreover, it is believed that the use of bismuth thiols together
with other biofilm-inhibiting- and/or wound healing agents can
provide synergistic benefits when treating chronic wounds and other
tissue defects. While not wishing to be bound by theory, it is
believed that the biofilm-inhibiting substances of the present
invention promote the wound-healing process by obviating the
negative consequences of wound bed biofilms on the wound- or
defect-healing process. It is further believed that when used in
combination with bioremodelable ECM materials, a further
enhancement in healing can be achieved.
In view of the advantageous properties of the medical products of
this invention, it is believed that treatment times can be reduced,
as well as the need for repeated debridement of the tissue defect
area.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic illustration of a medical device construct in
accordance with the present invention.
FIG. 2 is a schematic illustration of a medical device construct
according to another aspect of the present invention.
FIG. 3 is a schematic illustration of a medical device construct
according to further aspect of the present invention.
FIG. 4 is a schematic illustration of a medical device construct
according to yet another aspect of the present invention.
DETAILED DESCRIPTION
It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to limit the scope of the present invention which will be
limited only by the appended claims. It must be noted that as used
herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly
dictates otherwise. Thus, for example, reference to "a cell" is a
reference to one or more cells and includes equivalents thereof
known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices, and materials similar or equivalent to those
described herein may be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
Definitions of Terms
As used herein, the term "wound" denotes a bodily injury with
disruption of the normal integrity of tissue structures. The term
is also intended to encompass the terms "sore", "lesion",
"necrosis" and "ulcer". The term "sore" typically refers to any
lesion of the skin or mucous membranes. The term "ulcer" refers to
a local defect, or excavation, of the surface of an organ or
tissue, which is produced by the sloughing of necrotic tissue. The
term "lesion" relates to any tissue defect. The term "necrosis"
relates to dead tissue resulting from infection, injury,
inflammation or infarctions.
The term "chronic wound" denotes a wound not healed or typically
not healed after 4 to 6 weeks of conventional treatment.
The term "biofilm" denotes an extracellular matrix in which
microorganisms are dispersed and/or form colonies. The biofilm
typically is made of polysaccharides and other macromolecules. In
addition, in the present invention, the phrase "inhibiting a
biofilm," and like phrases, means the prevention of biofilm growth,
reduction in the rate of biofilm growth, partial eradication of
existing biofilm, and/or complete eradication of existing
biofilm.
The term "biocidal" is art recognized and includes broad spectrum
acting agents believed by those of ordinarily skill in the art
prior to the present invention to kill microbial cells
"non-specifically" by modes of action affecting a plurality of
different targets. Examples of biocidal agents include bismuth
thiols, paraben, chlorobutanol, phenol, alkylating agents such as
ethylene oxide and formaldehyde, halides, mercurials and other
heavy metals, detergents, acids, alkalis, disinfectants, pine oil,
triclosan, quaternary amine compounds such as alkyl dimethyl benzyl
ammonium chloride, chloroxylol, chlorhexidine, and cyclohexidine,
and triclocarbon.
The term "antibiotic" is art recognized and includes antimicrobial
agents synthesized by an organism, isolated from the natural
source, and includes natural or chemically synthesized analogs
thereof. The term includes but is not limited to: polyether
ionophore such as monensin and nigericin; macrolide antibiotics
such as erythromycin and tylosin; aminoglycoside antibiotics such
as streptomycin and kanamycin; beta-lactam antibiotics such as
penicillin and cephalosporin; and polypeptide antibiotics such as
subtilisin and neosporin. In contrast to the term "biocidal," an
antibiotic is considered to have a specific molecular target in a
microbial cell.
The term "bactericidal" refers to an agent that can kill a
bacterium; "bacteriostatic" refers to an agent that inhibits the
growth of a bacterium.
The term "quorum sensing signaling" or "quorum sensing" denotes
generation of a cellular signal in response to cell density. In one
embodiment, quorum sensing signaling mediates the coordinated
expression of specific genes.
The term "quorum sensing controlled gene" denotes a gene whose
expression is regulated in a cell density dependent fashion. A
quorum sensing controlled gene may encode a protein or polypeptide
that, either directly or indirectly, inhibits and/or antagonizes a
bacterial host defense mechanism or that regulates biofilm
formation.
The term "quorum sensing signal molecule" denotes a molecule that
transduces a quorum sensing signal and mediates the cellular
response to cell density. The quorum sensing signal molecule may
regulate expression of the quorum sensing controlled gene.
Exemplary quorum sensing signal molecules include freely diffusible
autoinducer molecules, such as homoserine lactone molecules or
analogs thereof.
The term "bioremodelable" refers to a natural or synthetic material
capable of inducing tissue remodeling in a subject or host. A
bioremodelable material can include at least one bioactive agent
(e.g., growth factor, etc.) capable of inducing tissue remodeling.
The ability to induce tissue remodeling may be ascribed to one or
more bioactive agents in a bioremodelable material stimulating the
infiltration of native cells into an a cellular matrix, stimulating
new blood vessel formation (capillaries) growing into the matrix to
nourish the infiltrating cells (angiogenesis), and/or effecting the
degradation and/or replacement of the bioremodelable material by
endogenous tissue. The bioremodelable material may include
extracellular collagen matrix (ECM) material, including but not
limited to submucosal tissue, such as small intestine submucosal
(SIS) tissue or it may include other natural tissue source
materials, or other natural or synthetic materials, including one
or more bioactive substances capable of inducing tissue
remodeling.
The terms "angiogenesis and angiogenic" refer to bioremodelable
properties defined by formation of capillaries or microvessels from
existing vasculature in a process necessary for tissue growth,
where the microvessels provide transport of oxygen and nutrients to
the developing tissues and remove waste products.
The term "submucosa" refers to a natural collagen-containing tissue
structure removed from a variety of sources including the
alimentary, respiratory, intestinal, urinary or genital tracts of
warm-blooded vertebrates. Submucosal material according to the
present invention includes tunica submucosa, but may include
additionally adjacent layers from the source tissue, such as the
lamina muscularis mucosa and the stratum compactum, the lamina
propria and/or other tissue structures. It will be understood that
the submucosal material may include all or only a portion of the
original tunica submucosa of a source tissue, considered in terms
of thickness and/or width of the original tunica submucosa. A
submucosal material may be a decellularized or acellular tissue,
which means it is devoid of intact viable cells, although some cell
components may remain in the tissue following purification from a
natural source. Alternative embodiments (e.g., fluidized
compositions etc.) include submucosal material expressly derived
from a purified submucosal matrix structure. Submucosal materials
according to the present disclosure are distinguished from collagen
materials in other medical devices that do not retain their native
submucosal structures or that were not prepared from purified
submucosal starting materials first removed from a natural
submucosal tissue source.
The term "small intestinal submucosa" (SIS) refers to a particular
type of submucosal structure removed from a small intestine source,
such as pig.
The term "biocompatible" refers to something, such as certain types
of extracellular matrix material, that can be substantially
non-toxic in the in vivo environment of its intended use, and is
not substantially rejected by the patient's physiological system
(i.e., is non-antigenic). This can be gauged by the ability of a
material to pass the biocompatibility tests set forth in
International Standards Organization (ISO) Standard No. 10993
and/or the U.S. Pharmacopeia (USP) 23 and/or the U.S. Food and Drug
Administration (FDA) blue book memorandum No. G95-1, entitled "Use
of International Standard ISO-10993, Biological Evaluation of
Medical Devices Part-1: Evaluation and Testing." Typically, these
tests measure a material's toxicity, infectivity, pyrogenicity,
irritation potential, reactivity, hemolytic activity,
carcinogenicity and/or immunogenicity. A biocompatible structure or
material, when introduced into a majority of patients, will not
cause a significantly adverse, long-lived or escalating biological
reaction or response, and is distinguished from a mild, transient
inflammation which typically accompanies surgery or implantation of
foreign objects into a living organism.
The term "therapeutically effective amount" refers to an amount of
a modulator, drug or other molecule that is sufficient to effect
treatment when administered to a subject in need of such treatment.
A therapeutically effective amount will vary depending upon the
subject and disease condition being treated, the weight and age of
the subject, the severity of the disease condition, the manner of
administration and the like.
The term "carrier" refers to a natural or synthetic material
carrying and delivering one or more exogenously added bioactive
agents, including bismuth thiol agents, such as
bismuth-1,2-ethandithiol; other biofilm-inhibiting agents, such as
lactoferrin, xylitol, quorum sensing inhibitors, biocidal agents,
antibiotics, and surfactants; wound healing agents, such as growth
factors, cytokines, and protease inhibitors; analgesic agents, and
the like.
The term "gel" refers to a three-dimensional polymer network that
includes a liquid solvent entrained by an interconnected matrix of
polymer chains. More particularly, the term refers to polymer
networks between the liquid and solid state containing enough
solvent molecules to cause macroscopic changes in the sample
dimension. The term is also meant to include gels in their "dry"
condition, in which all substantially all solvent that is within
the gel matrix has been removed. The term dry is primarily an
operational definition. One definition of the term is when the mass
of the gel reaches a constant low value in desiccator or drying
oven.
The terms "patient," "subject," and "recipient" as used in this
application refer to any mammal, especially humans. For purposes of
treatment, the term "mammal" refers to any animal classified as a
mammal, including humans, domestic and farm animals, and zoo,
sports, or pet animals, such as dogs, horses, cats, cattle, pigs,
sheep, etc. Preferably, the mammal is human.
In one aspect, a wound dressing, graft material or other medical
product includes a carrier formed from natural, bioremodelable
material, whereby the carrier further includes or is formulated to
deliver, a biocidal bismuth thiol agent and/or other
biofilm-inhibiting- or wound healing agents. In a preferred
embodiment the carrier is formed from extracellular matrix (ECM)
material, and the bismuth thiol is bismuth-1,2-ethanedithiol.
When applied to a wound, the dressing enhances wound treatment by
preventing or reducing biofilm formation or development. The
bioremodelable matrix provides a material source to promote wound
healing and tissue rebuilding. For example, when placed over a
wound, the bioremodelable ECM materials provide a rapidly
vascularized matrix to support the growth and remodeling of new
endogenous tissue, and the biofilm-inhibiting agents serve to
reduce or prevent biofilm formation and development to enhance
wound treatment.
Wound dressings or other medical products of the present invention
employ carriers which may be applied in a variety of forms,
including single- or multi-layer sheet constructs, fluidized
formulations, and/or combinations thereof. Sheet constructs or
fluidized formulations may be made from bioremodelable ECM-based
materials. The sheet or fluidized carrier materials may be further
formed into a suitable 3-D structure, such as a plug or wedge or
applied in a dried powdered form. A bismuth thiol containing ECM
material can be used in the treatment of a fistula, including a
fistula having an opening into the gastrointestinal tract such as
an anorectal fistula, enterocutaneous fistula, a rectovaginal
fistula, or others. For these purposes, the ECM material can be
processed into the form of a plug or other shape to occlude at
least the primary opening of the fistula, as disclosed for example
in United States Patent Application Publication No. 2008/0004657
dated 3 Jan. 2008 and United States Patent Application Publication
No. 2007/0088445 dated Apr. 19, 2007, each of which is hereby
incorporated herein by reference in its entirety.
Additional biocompatible substrate films or layers may be included
in conjunction with the carrier, including a top sheet to restrict
passage of liquid back towards the wound or defect; a backing layer
providing a barrier to passage of microorganisms through the
dressing; an absorbent layer for absorbing wound fluids; and/or an
adhesive layer forming an adhesive-coated margin.
Wound dressings of the present invention may be used to manage a
variety of wounds, including partial and full thickness wounds,
diabetic ulcers, venous ulcers, chronic vascular ulcers, leg
ulcers, pressure ulcers, decubitus, ulcus cruris,
tunneled/undermined wounds, fistulae, surgical wounds (such as
donor site wounds for autografts, post-Moh's surgery wounds,
post-laser surgery wounds, wound dehiscence), trauma wounds (such
as abrasions, lacerations, second-degree burns, and skin tears),
and draining wounds.
Graft materials or graft products of the invention can find wide
use in the field of medicine, and in this regard, can be adapted to
provide a variety of devices and objects suitable for application
to and/or implantation within a patient. The present invention also
provides, in certain aspects, various methods for using these
materials, for example, to replace, augment, repair, and/or
otherwise suitably treat diseased or otherwise damaged or defective
tissue of a patient. Illustratively, graft materials of the
invention can be configured as implantable devices suitable for
bulking tissue, providing hemostasis, and/or providing occlusion in
a passageway or other open space within the body of a patient
(e.g., as an embolization device, fistula plug, etc.). In some
embodiments, graft materials of the invention are configured as
single- or multilayered patches or other sheet or sheet-like
devices for providing support to patient tissue or otherwise
treating patient tissue. Illustratively, inventive graft materials
can provide wound healing products suitable for cutaneous,
intracutaneous, and/or subcutaneous wound treatment, e.g., a hernia
repair patch or a burn treatment material. As well, sheet-form
graft products of the invention find use as precursor materials for
forming a variety of other medical devices, or components thereof.
Illustratively, graft materials of the invention can be processed
into various shapes and configurations, for example, into a
urethral sling or a prosthetic body part. In some forms, sheet-form
graft materials of the invention are suitable for forming tubular
grafting devices, which may be used to replace a circulation
vessel, or a portion thereof, or to bypass a blocked vessel.
In one preferred embodiment, medical products, including graft
materials in accordance with the present invention can be used as
tissue grafts in mammalian patients, including humans. Graft
materials in accord with certain embodiments of the invention can
be useful in the repair or support of soft tissue areas, such as
body walls. The graft products or graft materials can include any
of the carrier formulations or medical device constructs described
herein for internal or external grafting. For example, in
illustrative embodiments, graft materials of the invention can be
used in hernia repair applications. Such applications include the
repair of a hiatal hernia by affixing or securing the graft product
over or near a tear in the esophageal hiatus of the diaphragm. The
graft product can be fastened or secured to the esophageal hiatus
by placing sutures and/or staples, and/or other suitable fasteners
and/or the like, through the reinforcement bands in the graft
product and into the esophageal hiatus.
In alternative embodiments, graft products of the invention can be
used in the repair or support of tissue surrounding an inguinal
hernia by affixing a graft product of this invention over and/or
near to a tear in the abdominal wall in the groin region. Again,
the graft product can be attached to the soft tissue of the
abdominal wall by placing sutures, and/or staples, and/or other
fasteners through the reinforcement band or bands present in the
graft product and into the tissue wall. In addition to repairing
inguinal hernias, the present invention may also include
implantable grafts suitable for other types of body wall
reinforcement or repair. Further examples of implantable grafts
include slings configured to prevent prolapse of a particular
pelvic organ, such as the urethra, bladder, rectum, or small
bowel.
As indicated above, wound dressings, graft materials or other
medical products of the present invention include a biocidal
bismuth thiol agent and a carrier. The carrier includes a natural,
bioremodelable material, preferably an extracellular matrix (ECM)
material, such as submucosal tissue material in solid or fluidized
form.
FIG. 1 is a schematic illustration of medical device construct or
medical device 10 (such as wound dressing) including an ECM
material layer 12, where bismuth thiols 14 are applied by coating
onto at least one side of the ECM material layer 12. The medical
device construct 10 may be applied to a tissue directly or it may
be attached to or incorporated into another medical device or
product.
The carrier may further include other biofilm-inhibiting agents,
wound healing agents, and/or analgesic agents. One or more of these
agents may be exogenously incorporated into the carrier during
their preparation or covalently attached to the carrier when
preparing the wound dressing or other medical product.
Alternatively, the biofilm-inhibiting and/or wound healing agents
may be added to the carrier after preparation of the carrier, e.g.,
by coating, soaking, spraying, painting, or otherwise applying the
biofilm-inhibiting and/or wound healing agent(s) to the
carrier.
In another aspect, the carrier includes fluidized ECM materials
formulated as a substantially homogenous biofilm-inhibiting
solution containing bismuth thiols and/or other exogenously added
bioactive agents. Fluidized ECM materials may be dried or formed
into a gel or foam for direct application to a wound-contacting
layer or tissue defect further utilized in a composite wound
dressing including one or more biocompatible substrate layers. The
resulting multilayer construct may be dried by lyophilization.
FIG. 2 is a schematic illustration of an exemplary medical device
construct 20 including an ECM material layer 22 where bismuth
thiols 24 are incorporated into the ECM material layer 22 by mixing
the bismuth thiols 24 into a fluidized ECM solution and allowing
the resultant solution to dry in the form of a dried cake 26. The
dried cake 26 may be used as a medical device (e.g. a wound
dressing) by itself or it may be attached or incorporated into
another medical device or product. Alternatively, other
biofilm-inhibiting agents may be additionally added to aid in the
repair, replacement, treatment, and/or healing of a wound. The
additional biofilm-inhibiting agents may be added to the surface of
the dried cake 26 as a layer 28 or they may be incorporated into
the fluidized ECM solution prior to forming the dried cake 26.
FIG. 3 is a schematic illustration of an exemplary medical device
construct 30, including an ECM material layer formed from a
fluidized ECM solution, dried into a cake 36 and adhered to a
biocompatible base substrate layer 39. In a preferred embodiment,
the biocompatible base substrate layer 39 comprises a sheet
containing at least one natural layer of extracellular matrix
material.
FIG. 4 depicts an alternative medical device construct 40 in which
a dried ECM-based cake layer 46 is sandwiched between two
biocompatible base substrate layers 49. In a preferred embodiment,
each biocompatible base substrate layer 49 comprises a sheet of
extracellular matrix material.
The ECM-based cake layers can have any thickness desired.
Generally, the thickness of this layer in certain embodiments will
be from about 10 microns to about 10 mm, more typically about 0.1
mm to about 5 mm. A dried ECM-based cake layer will typically have
a more open structure than the underlying base sheet material and
will also in advantageous embodiments be less dense and/or less
strong under tension than the underlying base or sheet material.
Preferred dried cake layers will have a somewhat spongy character
when dry.
The dried ECM-based cake may be subjected to further processing if
desired, including for example cross-linking with any suitable
agent such as radiation, chemical agents, or the like. In certain
embodiments, treatment to cross-link the cake or layer is not
performed (i.e. no additional cross-linking is introduced into the
fibrillar mass), and in such embodiments desirable biotropic
properties, including angiogenic properties, of the deposited layer
can be retained. In other embodiments, cross-linking may be
undertaken, but to an extent wherein the deposited cake retains
bioactive (e.g. angiogenic) properties. These and other variations
in processing of the deposited cake will occur to the skilled
artisan in view of the teachings herein.
The drying of the deposited gel or other liquid-containing
composition to form a dried cake or mass may be conducted in any
suitable fashion. Preferably, the drying is conducted by a
lyophilization technique, including for example a lyophilization
technique involving freeze-drying and/or evaporative cooling. Other
drying techniques such as air drying, drying under heated
conditions, or vacuum pressing, may also be used to provide all or
portion of the drying function.
In one aspect, a bioremodelable ECM material (sheet or fluidized)
is adhered to a biocompatible base substrate in which the base
substrate includes or is made from an ECM sheet material. An
exemplary ECM sheet material is a sheet of submucosa tissue graft
material (OASIS.RTM. Wound Matrix, Cook Biotech Incorporated, West
Lafayette, Ind., USA).
When attached to a biocompatible substrate layer, the fluidized ECM
material constitutes a biotropic mass of submucosa-derived
components adhered to at least one substrate layer surface. A dried
or gelled layer of fluidized ECM may be sandwiched or otherwise
positioned between two substrate layers. Alternatively, one or more
dried or gelled layers of fluidized ECM may be adhered to one or
more base substrate layers.
A wide variety of biocompatible base substrate materials may be
used. Exemplary base substrate materials include sheet or other
substrate materials comprised of biopolymers such as collagen or
gelatin, as well as sheet or other substrate materials made from
synthetic polymers, resorbable and/or non-resorbable. Substrate
materials made with combinations of biopolymers and synthetic
polymers are also suitable for use in the invention. In a preferred
embodiment, the substrate material is bioremodelable. An exemplary
substrate material is a sheet of submucosa tissue graft material
(OASIS.RTM. Wound Matrix, Cook Biotech Incorporated, West
Lafayette, Ind., USA).
The biocompatible base substrate may be formed as a monolayer.
Alternatively, a variety of techniques for laminating materials,
including ECMs, together are known and may be used to prepare
multilaminate base substrates. For example, a plurality of (i.e.
two or more) layers of collagenous material, for example
submucosa-containing or other ECM material, may be bonded together
to form a multilaminate structure. Illustratively, two, three,
four, five, six, seven, or eight or more collagenous layers
containing submucosal or other collagenous ECM materials may be
bonded together to provide a multilaminate collagenous substrate
material for use in the present invention. In certain embodiments,
two to six collagenous, submucosa-containing layers isolated from
intestinal tissue of a warm-blooded vertebrate, particularly small
intestinal tissue, are bonded together. Porcine-derived small
intestinal tissue is preferred for this purpose. The layers of
collagenous tissue may be bonded together in any suitable fashion,
including dehydrothermal bonding under heated, non-heated or
lyophilization conditions, using adhesives, glues or other bonding
agents, crosslinking with chemical agents or radiation (including
UV radiation), or any combination of these with each other or other
suitable methods.
In one aspect, one or more layers of ECM sheet material (monolayer
or multilaminate) containing bismuth thiols with or without other
bioactive agents may be directly applied to the wound bed.
Secondary dressing materials may be applied over the sheet of ECM
material to keep the wound moist and to allow the flow and
absorption of wound exudates. Suitable secondary dressing materials
include Adaptic.RTM. Dressing (Ethicon Inc., Somerville, N.J.,
USA), DuoDERM CGF Control Gel Formula Dressings (ConvaTec, A
Bristol Myers Suibb Company, Princeton, N.J., USA) and various
others known to those of skill in the art.
Alternatively, the ECM materials may be adhesively secured to a
wound area or other tissue defect by coating one or more skin or
other tissue contacting surfaces of the medical device construct or
medical product with a suitable adhesive. Suitable adhesives
include pressure-sensitive adhesives. The adhesive may be attached
to one or more release sheets to provide aseptic protection for the
coated front surface of the wound-contacting surface of the
dressing and to facilitate precise positioning of the dressing over
the wound or other defect. Suitable adhesives and release (or
attachment) sheets are described in U.S. Pat. No. 5,052,281, which
is incorporated by reference herein.
In another aspect, an ECM gel layer may be disposed between a
wound-contacting or defect-contacting film or sheet and an
absorbent layer. Such a configuration may provide a more moist
wound/defect environment for prolonged periods. Since fluidized
gels are known to be useful in providing controlled release of
bioactive agents, biofilm-inhibiting agents, wound healing agents
and the like may be incorporated in the fluidized gel formulations
for delivery to the wound bed.
In another aspect, a bioremodelable ECM gel layer is adhered to a
top sheet film, which is formulated to allow fluid from the wound
or defect to pass through the sheet toward the ECM gel layer, but
to restrict the passage of liquid back towards the wound or defect.
Preferably the wound/defect facing surface of the top sheet film is
made hydrophobic so as to reduce adherency of the top sheet to the
wound or other defect.
The wound or defect contacting sheet may include a top sheet film
formed from a thermoplastic film-forming polymer. Preferably, the
polymer is conformable but not substantially elastomeric. Exemplary
polymers include, but are not limited to, polyethylene,
polypropylene, polyester, polyamides such as nylons, fluoropolymers
such as polyvinylidene fluoride (PVDF) or polytetrafluoroethylene
(PTFE), and mixtures thereof. The top sheet is preferably a
polyolefin film. Preferably, the film has a thickness by weight
(ASTM E252-84) of from 10 to 200 micrometers, more preferably from
25 to 100 micrometers.
Additionally, the top sheet is preferably formed from a
substantially liquid-impermeable sheet material provided with
tapered capillaries, each capillary having a base substantially in
the plane of the wound or defect facing surface of the top sheet
and an apical opening remote from the wound or defect facing
surface of the top sheet and preferably in contact with the gel
and/or the absorbent layer. The conical capillaries provide rapid
one-way wicking of fluid from the front of the top sheet, with
minimal wet-back (i.e. back flow of fluid toward wound). Top sheets
of the above type are described in GB-A-1526778 and US
2005/0256437.
In a further aspect, an ECM material (sheet or gel) is adhered to a
second biocompatible base substrate in the form of an absorbent
layer or wicking layer to absorb fluids and exudate from the wound
or other defect. The absorbent or wicking layer may be formed from
one or more layers or plies of the same or different absorbent
materials. Preferably, the wicking layer is substantially the same
size and shape as the wound/defect-facing layer, or slightly
smaller than the wound/defect-facing layer.
The absorbent layer or wicking layer is preferably formed from any
one of a variety of conventional materials for absorbing wound
exudates, fluids, serum, or blood known in the wound healing art,
including thermoplastic, water-swellable polymer films; absorbent
foams; hydrogel materials; gauzes; nonwoven, woven, and knitted
fabrics; superabsorbents; and mixtures thereof. The absorbent layer
may be made from any of the absorbent materials described in U.S.
Pat. Nos. 5,981,822 and 6,566,577, which are incorporated by
reference herein.
The ECM material layer or the absorbent layer may be further
adhered to a backing sheet layer formed from a breathable,
substantially microbe-impermeable, liquid impermeable material or
film configured to protect the wound or defect from further
microbial infection or contamination and to prevent or reduce
leakage of wound or defect exudate into clothes, bedclothes, etc.
Suitable backing sheet layers and backing layer materials are
described in U.S. Pat. No. 6,566,577, which is incorporated by
reference herein.
Wound dressings or other medical products of the present invention
may further comprise one or more protective cover sheets over any
exposed surface or surface adhesive. For example, the protective
cover sheets may include one or more release-coated paper cover
sheets. Preferably, the wound dressing or other product is sterile
and packaged in a microorganism-impermeable container.
Wound dressings or other products may be supplied in standard
configurations suitable for application to a variety of wounds or
defects and may be applied as is or may be cut, molded or otherwise
shaped prior to application to a particular application site.
Alternatively, the wound dressings or other medical products may be
configured for a specific wound or specific wound or defect type.
Wound dressings may be adapted for localized wound applications or
as whole wound dressings. In certain embodiments, the medical
product is sized, shaped and exhibits sufficient strength to treat
a hernia. In other embodiments, the medical product is formed into
a plug or other shape and used to treat a fistula.
A wound dressing or other medical product may be supplied and/or
applied to a wound or defect as moist material ready for
application to a wound or defect area or it may be supplied and/or
applied as a dried material that can be rehydrated (with saline,
for example) upon or prior to application to a wound or defect
area. In some embodiments, a biological glue or tissue adhesive may
be provided between a debrided wound bed surface and a contacting
layer or sheet of ECM material to hold the ECM material in a
stationary position against the wound bed surface.
An exemplary tissue adhesive is BioGlue.RTM. (CryoLife, Inc.).
Other suitable adhesives include fibrin-, fibrinogen-, and
thrombin-based sealants, bioactive ceramic-based sealants, and
cyanoacrylate sealants, including, but not limited to, Vitex (V.I.
Technologies, NY; comprising thrombin:fibrinogen in a 1:1 ratio);
Quixil (Omrix Biopharm SA, Brussels); Dermabond, an
octylcyanoacrylate tissue adhesive (Bruns and Worthington (2000)
Am. Fam. Physician 61:1383-1388); Tisseel (Baxter International,
Deerfield, Ill.); Hemaseel APR (Haemacure, Sarasota, Fla.);
PlasmaSeal (Plasmaseal, San Francisco, Calif.); AutoSeal (Harvest
Technologies, Norwell, Mass.); Floseal (Fusion Medical
Technologies, Mountain View, Calif.); and Bioglass (U.S.
Biomaterials, Alachua, Fla.); CoStasis (Cohesion Technologies). Med
Pro Month (1999) 9:261-262; and MedPro Month (2000) 10:86-91.
The tissue adhesive may be bioresorbable. A bioresorbable adhesive
may be formed by forming intermacromolecular complexes of a
carboxypolysaccharide and, optionally, a polyether, such as
polyethylene oxide. The carboxypolysaccharide may be of any
biocompatible sort, including but not limited to carboxymethyl
cellulose (CMC), carboxyethyl cellulose, chitin, hyaluronic acid,
starch, glycogen, alginate, pectin, carboxymethyl dextran,
carboxymethyl chitosan, and glycosaminoglycans such as heparin,
heparin sulfate, and chondroitin sulfate. It is within the scope of
this disclosure, however, to include any type of tissue adhesive
sufficient for adhering ECM materials to the wound bed surface.
The wound dressings disclosed herein may be used to create
bioresorbable wound dressings or band-aids. Wound dressings may be
used as a wound-healing dressing, a tissue sealant (i.e., sealing a
tissue or organ to prevent exposure to a fluid or gas, such as
blood, urine, air, etc., from or into a tissue or organ), and/or a
cell-growth scaffold. In various embodiments, the wound dressing
may protect the injured tissue, maintain a moist environment, be
water permeable, be easy to apply, not require frequent changes, be
non-toxic, be non-antigenic, maintain microbial control, and/or may
deliver effective healing agents to the wound site.
Examples of bioresorbable sealants and adhesives that may be used
in accordance with the wound dressing described herein include, for
example, FOCALSEAL.RTM. (biodegradable eosin-PEG-lactide hydrogel
requiring photopolymerization with Xenon light wand) produced by
Focal; BERIPLAST.RTM. produced by Adventis-Bering; VIVOSTAT.RTM.
produced by ConvaTec (Bristol-Meyers-Squibb); SEALAGEN.TM. produced
by Baxter; FIBRX.RTM. (containing virally inactivated human
fibrinogen and inhibited-human thrombin) produced by CyoLife;
TISSEEL.RTM. (fibrin glue composed of plasma derivatives from the
last stages in the natural coagulation pathway where soluble
fibrinogen is converted into a solid fibrin) and TISSUCOL.RTM.
produced by Baxter; QUIXIL.RTM. (Biological Active Component and
Thrombin) produced by Omrix Biopharm; a PEG-collagen conjugate
produced by Cohesion (Collagen); HYSTOACRYL.RTM. BLUE (ENBUCRILATE)
(cyanoacrylate) produced by Davis & Geek; NEXACRYL.TM. (N-butyl
cyanoacrylate), NEXABOND.TM., NEXABOND.TM. S/C, and TRAUMASEAL.TM.
(product based on cyanoacrylate) produced by Closure Medical
(TriPoint Medical); DERMABOND.TM. which consists of 2-Octyl
Cyanoacrylate produced by Dermabond (Ethicon); TISSUEGLU.RTM.
produced by Medi-West Pharma; and VETBOND.TM. which consists of
n-butyl cyanoacrylate produced by 3M.
Natural, Bioremodelable ECM Source Materials
In accordance with the present invention, the wound dressing
includes a carrier. In one aspect, the carrier is formed from
natural, bioremodelable materials. In a preferred embodiment, the
carrier is formed from extracellular matrix (ECM) material(s),
which are preferably bioremodelable. Upon application of the wound
dressing to the body of a subject, the bioremodelable material in
the wound dressing may serve as a matrix to promote and/or induce
the growth and/or bioremodeling of endogenous tissue. These are
properties particularly suited to meet the requirements for healing
wounds and other tissue defects. Bioremodelable materials of the
present invention are preferentially selected to facilitate the
process of bioremodeling, which may include: (1) stimulation in the
infiltration of native cells into an acellular matrix; (2)
stimulation of new blood vessel formation (capillaries) growing
into the matrix to nourish the infiltrating cells (angiogenesis);
and/or (3) effecting the degradation and/or replacement of the
bioremodelable material by endogenous tissue upon implantation into
a host.
Bioremodelable materials have been used successfully in vascular
grafts, urinary bladder and hernia repair, replacement and repair
of tendons and ligaments, and dermal grafts. When used in such
applications, the graft constructs appear not only to serve as a
matrix for the regrowth of the tissues replaced by the graft
constructs, but also to promote or induce such regrowth of
endogenous tissue. Common events in the remodeling process include
widespread, rapid neovascularization, proliferation of granulation
mesenchymal cells, biodegradation/resorption of implanted
intestinal submucosal tissue material, and lack of immune
rejection. When positioned in a wound area, the wound dressing is
capable of being ultimately replaced by endogenous host
tissues.
Bioremodelable ECM materials for use in the present invention may
possess one or more angiogenic properties. Angiogenesis represents
a crucial step in tissue formation in response to biomaterial
implantation, especially necessary for implants that are designed
to foster tissue growth. Angiogenesis is a complex process that
depends on many mechanisms occurring in an organized manner (P.
Carmeliet, Mechanisms of angiogenesis and arteriogenesis, Nat Med 6
(2000), no. 4, 389-395). Due to the complexity necessary for proper
angiogenesis, biomaterial interaction with the host environment can
have a dramatic effect on the quality and quantity of the
angiogenic activity. Methods for measuring in vivo angiogenesis in
response to biomaterial implantation have recently been developed.
One such method uses a mouse subcutaneous implant model to
determine the angiogenic potential (Heeschen, C. et al., Nat Med
vol. 7, no. 7, pp. 833-839, 2001). When combined with a
fluorescence microangiography technique (Johnson, C. et al., Circ
Res., vol. 94, no. 2, pp. 262-268, 2004), this model can give
quantitative and qualitative measures of angiogenesis into
biomaterials.
Bioremodelable materials may include naturally-derived collagenous
ECM materials isolated from suitable animal or human tissue
sources. As used herein, it is within the definition of a
"naturally-derived ECM" to clean, delaminate, and/or comminute the
ECM, or to cross-link the collagen or other components within the
ECM. It is also within the definition of naturally occurring ECM to
fully or partially remove one or more components or subcomponents
of the naturally occurring matrix.
Bioremodelable ECM materials possess biotropic properties capable
of inducing tissue remodeling. Suitable ECM materials which can be
processed to provide bioremodelable materials include, for example,
submucosal (including for example small intestinal submucosa (SIS),
stomach submucosa, urinary bladder submucosa, or uterine submucosa,
each of these isolated from juvenile or adult animals), renal
capsule membrane, dermal collagen, amnion, dura mater, pericardium,
serosa, peritoneum or basement membrane layers or materials,
including liver basement membrane or epithelial basement membrane
materials.
Submucosal tissue materials may be isolated and used as intact
natural sheet forms, as reconstituted collagen layers including
collagen derived from these materials, or as fluidized submucosal
solutions configured in the form of a gel, foam or a sponge. For
additional information as to submucosa materials useful in the
present invention, and their isolation and treatment, reference can
be made to U.S. Pat. Nos. 4,902,508, 5,554,389, 5,733,337,
5,993,844, 6,206,931, 6,099,567, and 6,331,319. Renal capsule
membrane can also be obtained from warm-blooded vertebrates, as
described more particularly in International Patent Application
serial No. PCT/US02/20499, published as WO 03002165. Commercially
available ECM materials capable of remodeling to the qualities of
its host when implanted in human soft tissues include porcine SIS
material (Surgisis.RTM. line of SIS materials, Cook Biotech Inc.,
West Lafayette, Ind.) and bovine pericardium (Peri-Strips.RTM.,
Synovis Surgical Innovations, St. Paul, Minn.).
The following U.S. patents, hereby incorporated by reference,
disclose the use of ECMs for the regeneration and/or repair of
various tissues: U.S. Pat. Nos. 6,379,710; 6,187,039; 6,176,880;
6,126,686; 6,099,567; 6,096,347; 5,997,575; 5,993,844; 5,968,096;
5,955,110; 5,922,028; 5,885,619; 5,788,625; 5,762,966; 5,755,791;
5,753,267; 5,733,337; 5,711,969; 5,645,860; 5,641,518; 5,554,389;
5,516,533; 5,460,962; 5,445,833; 5,372,821; 5,352,463; 5,281,422;
and 5,275,826.
Preferred ECM materials contain residual bioactive proteins or
other ECM components derived from the tissue source of the
materials. For example, they may contain fibroblast growth factor 2
(basic FGF), vascular endothelial growth factor (VEGF),
transforming growth factor-beta (TFG-beta), epidermal growth factor
(EGF), platelet-derived growth factor (PDGF), and/or isoforms or
combinations thereof. It is also expected that ECM base materials
of the invention may contain additional residual bioactive agents
including, for example, one or more of glycosaminoglycans,
glycoproteins, proteoglycans, and/or growth factors.
Further, in addition or as an alternative to the inclusion of
native bioactive agents, non-native bioactive agents such as those
synthetically produced by recombinant technology or other methods,
may be incorporated into the ECM materials of the present
invention. These non-native bioactive agents may be
naturally-derived or recombinantly produced proteins that
correspond to those natively occurring in the ECM tissue, but
perhaps of a different species (e.g. human proteins applied to
collagenous ECMs from other animals, such as pigs). The non-native
bioactive substances may be applied to the ECM material during its
preparation, prior to wound dressing application or it may be
applied during or after engraftment of the ECM material in the
patient. Exemplary non-native bioactive agents include growth
factors, cytokines, protease inhibitors, and the like.
Other non-native bioactive agents that may be incorporated into the
ECM materials or used in conjunction with the wound dressings of
the present invention include any of the above described
biofilm-inhibiting agents or bioactive agents, which are not
ordinarily present in ECM materials, including but not limited to
and analgesic agents, and the like.
Submucosal materials, including SIS materials, represent preferred
examples of ECM materials for use with the present invention. The
ECM materials may include residual bioactive proteins or other ECM
components derived from the tissue source of the materials. The ECM
materials may include (among others) fibroblast growth factor 2
(FGF-2), vascular endothelial growth factor (VEGF), transforming
growth factor-beta (TGF-beta). It is also expected that ECM base
materials of the invention may contain additional bioactive agents
including, for example, one or more highly conserved collagens,
growth factors, glycoproteins, proteoglycans, glycosaminoglycans,
other growth factors, and other biological materials such as
heparin, heparin sulfate, hyaluronic acid, fibronectin and the
like. Thus, generally speaking, submucosal or other ECM materials
may include a bioactive agent capable of inducing, directly or
indirectly, a bioremodeling response reflected in a change in cell
morphology, proliferation, growth, protein expression and/or gene
expression. The bioactive agents in the ECM materials may be
contained in their natural configuration and natural
concentration.
ECM or submucosal materials may be isolated from warm-blooded
vertebrate tissues including the alimentary, respiratory,
intestinal, urinary or genital tracts of warm-blooded vertebrates.
Preferred submucosal tissues may include intestinal submucosa,
stomach submucosa, urinary bladder submucosa, and uterine
submucosa. Intestinal submucosal tissue is one preferred starting
material, and more particularly intestinal submucosa delaminated
from both the tunica muscularis and at least the tunica mucosa of
warm-blooded vertebrate intestine.
An exemplary submucosa material is small intestine submucosa (SIS).
SIS has been shown to be acellular, strong, and exhibit a sidedness
in that it has a differential porosity of its mucosal and serosal
sides. Highly purified SIS generally does not trigger any negative
immune system responses, generally is free of viral activity, and
is known to reduce seepage. A preferred intestinal submucosal
tissue source in accordance with the present invention is porcine
SIS.
The preparation of intestinal submucosa is described in U.S. Pat.
Nos. 6,206,931 and 6,358,284, the disclosures of which are
incorporated by reference in their entirety herein. Urinary bladder
submucosa and its preparation are described in U.S. Pat. No.
5,554,389, the disclosure of which is incorporated by reference in
its entirety herein. Stomach submucosa and its preparation are
described in U.S. Pat. No. 6,099,567, the disclosure of which is
incorporated by reference in its entirety herein.
Preferred SIS material typically includes the tunica submucosa
delaminated from both the tunica muscularis and at least the
luminal portions of the tunica mucosa. The submucosal tissue may
include the tunica submucosa and basilar portions of the tunica
mucosa including the lamina muscularis mucosa and the stratum
compactum. The preparation of intestinal submucosa is described in
U.S. Pat. No. 4,902,508, and the preparation of tela submucosa are
described in U.S. Pat. Nos. 6,206,931 and 6,358,284, all of which
are incorporated by reference herein. The preparation of submucosa
is also described in U.S. Pat. No. 5,733,337, Nature Biotechnology,
vol. 17, p. 1083 (November 1999), and WO 98/22158. Also, a method
for obtaining a highly pure, delaminated submucosa collagen matrix
in a substantially sterile state was previously described in U.S.
Pat. Pub. No. 2004/180042, which is incorporated by reference
herein.
One preferred purification process involves disinfecting the
submucosal tissue source, followed by removal of a purified matrix
including the submucosa. It is thought that delaminating the
disinfected submucosal tissue from the tunica muscularis and the
tunica mucosa minimizes exposure of the submucosa to bacteria and
other contaminants following delamination and better preserves the
aseptic state and inherent biochemical form of the submucosa,
thereby potentiating its beneficial effects. Alternatively, the
ECM- or submucosa may be purified a process in which the
sterilization step is carried out after delamination as described
in U.S. Pat. Nos. 5,993,844 and 6,572,650. Still further preferred
processes for preparing an SIS or other ECM so as to provide
enhanced component profiles are described in U.S. Patent
Application Ser. No. 60/853,584 filed Oct. 23, 2006 and
International Application No. PCT/US2007/82238 filed Oct. 23, 2007,
each of which is hereby incorporated by reference in its entirety.
Accordingly, in certain embodiments, the ECM material retains
collagen and non-collagen components, and desirably exhibits an
angiogenic character. At the same time, the submucosa-containing or
other ECM material has low levels of undesired components such as
native lipids, nucleic acids (e.g. DNA), and/or immunoglobulin A
(IgA) components. In some embodiments, the ECM can be a sterile,
decellularized extracellular matrix (ECM) material including native
fibroblast growth factor-2 (FGF-2), and native immunoglobulin A
(IgA) at a level of no greater than 20 .mu.g/g. In some forms, this
ECM material can have a lipid content of no greater than about 4%.
In still further aspects, the ECM material can have a native FGF-2
content of at least about 10 ng/g and at least one of, and in
certain forms each of (i) native IgA at a level of no greater than
about 20 .mu.g/g; (ii) native lipids at a level of no greater than
about 4% by weight; (iii); (iv) native hyaluronic acid at a level
of at least about 50 .mu.g/g; and (v) native sulfated
glycosaminoglycan at a level of at least about 500 .mu.g/g. These
unique ECM materials can be prepared by processing methods that
comprise treating a relatively impure ECM starting material to
decrease the content of the undesired components, such as nucleic
acid, lipids and/or immunoglobulins such as IgA, while retaining
substantial levels of desired components such as growth factor(s),
proteoglycans and/or glycosaminoglycans (GAGs). Typically, to
prepare such preferred ECM materials, an ECM starting material will
be treated with a mild detergent solution, such as an ionic or
nonionic detergent solution. The low concentration of detergent
enables a retention of a substantial level of desired components,
such as those as noted above. In certain modes of operation, the
ECM material will be treated with an aqueous solution of sodium
dodecyl sulfate (SDS) or another ionic or nonionic detergent at a
detergent concentration of about 0.05% to about 1%, more preferably
about 0.05% to about 0.3%. This treatment can be for a period of
time effective to disrupt cell and nuclear membranes and to reduce
the immunoglobulin (e.g. IgA) content of the ECM material,
typically in the range of about 0.1 hour to about 10 hours, more
typically in the range of about 0.5 hours to about 2 hours.
Processing the isolated ECM material in this manner preferably
disrupts cell and nuclear membranes and results in a material with
a substantially reduced its IgA content, thus reducing the
immunogenicity of the material. In addition to treating an ECM
material with a detergent medium, the ECM material can be contacted
with other agents that participate in achieving the desired ECM
component profile. For example, the ECM material can be treated
with an aqueous medium, preferably basic, in which DNA is soluble.
Such a medium can in certain forms have a pH in the range of above
7 to about 9, with pH's in the range of about 8 to about 8.5
proving particularly beneficial in some embodiments. The basic
aqueous medium can include a buffer, desirably a biocompatible
buffer such as tris(hydroxymethyl)aminomethane (TRIS), and/or a
chelating agent such as ethylene diamine tetraacetic acid (EDTA).
In one preferred form, the nucleic acid solubilizing medium is a
TRIS-borate-EDTA (TBE) buffer solution. This treatment with a DNA
solubilizing medium can be for a period of time effective to reduce
the DNA content of the ECM material, typically in the range of
about 0.1 hour to about 10 hours, more typically in the range of
about 0.5 hours to about 2 hours. In addition to treatment with
detergent and DNA-solubilization media, methods of preparing
medical graft materials of the invention can involve treatment with
a liquid medium that results in a substantial reduction of the
level of lipid components of the ECM material. For example, the
resulting native lipid content of the ECM material can be reduced
to no greater than about 4% in certain embodiments. This can be
accomplished, for example, by a preparative process that involves a
step of treating the ECM material with a liquid organic solvent in
which the lipids are soluble. Suitable such organic solvents
include for example water-miscible solvents, including polar
organic solvents. These include low molecular weight (e.g. C.sub.1
to C.sub.4) alcohols, e.g. methanol, ethanol, isopropanol, and
butanols, acetone, chloroform, and others. This treatment with a
lipid-removing medium can be for a period of time effective to
reduce the lipid content of the ECM material, typically in the
range of about 0.1 hour to about 10 hours, more typically in the
range of about 0.1 hours to about 1 hours. In certain embodiments,
multiple (two or more) such treatments will be conducted.
The stripping of the submucosal tissue source is preferably carried
out by utilizing a disinfected or sterile casing machine, to
produce submucosa, which is substantially sterile and which has
been minimally processed. A suitable casing machine is the Model
3-U-400 Stridhs Universal Machine for Hog Casing, commercially
available from the AB Stridhs Maskiner, Gotoborg, Sweden. As a
result of this process, the measured bioburden levels may be
minimal or substantially zero. Other means for delaminating the
submucosa source can be employed, including, for example,
delaminating by hand.
In this method, a segment of vertebrate intestine, preferably
harvested from porcine, ovine or bovine species, may first be
subjected to gentle abrasion using a longitudinal wiping motion to
remove both the outer layers, identified as the tunica serosa and
the tunica muscularis, and the innermost layer, i.e., the luminal
portions of the tunica mucosa. The submucosal tissue is rinsed with
water or saline, optionally sterilized, and can be stored in a
hydrated or dehydrated state. Delamination of the tunica submucosa
from both the tunica muscularis and at least the luminal portions
of the tunica mucosa and rinsing of the submucosa provide an
acellular matrix designated as submucosal tissue. The use and
manipulation of such material for the formation of ligament and
tendon grafts and the use more generally of such submucosal tissue
constructs for inducing growth of endogenous connective tissues is
described and claimed in U.S. Pat. No. 5,281,422, disclosure of
which is incorporated herein by reference.
Following delamination, submucosa may be sterilized using any
conventional sterilization technique including glutaraldehyde,
formaldehyde, acidic pH, propylene oxide, ethylene oxide, gas
plasma sterilization, electron beam (E-beam) or other radiation
sterilization or peracetic acid sterilization, or combinations
thereof, and the like. Sterilization techniques which do not
adversely affect the mechanical strength, structure, and biotropic
properties of the purified submucosa are preferred. Certain
preferred sterilization techniques also include exposing the graft
to ethylene oxide treatment or gas plasma sterilization. Typically,
the purified submucosa is subjected to two or more sterilization
processes. After the purified submucosa is sterilized, for example
by chemical treatment, the matrix structure may be wrapped in a
plastic or foil wrap and sterilized again using electron beam or
gamma irradiation sterilization techniques. Certain sterilization
techniques may be found to be more compatible with a particular
bismuth thiol coated medical product. It will be understood that in
such instances, the sterilization technique(s) that are found to be
most compatible can be used.
Preferred submucosa may be characterized by the low contaminant
levels set forth in Table 1 below. The contaminant levels in Table
1 may be found individually or in any combination in a given ECM
sample. The abbreviations in Table 1 are as follows: CFU/g=colony
forming units per gram; PFU/g=plaque forming units per gram;
.mu.g/mg=micrograms per milligram; ppm/kg=parts per million per
kilogram.
TABLE-US-00001 TABLE 1 First Preferred Second Preferred Third
Preferred Level Level Level ENDOTOXIN <12 EU/g <10 EU/g <5
EU/g BIOBURDEN <2 CFU/g <1 CFU/g <0.5 CFU/g FUNGUS <2
CFU/g <1 CFU/g <0.5 CFU/g NUCLEIC <10 .mu.g/mg <5
.mu.g/mg <2 .mu.g/mg ACID VIRUS <500 PFU/g <50 PFU/g <5
PFU/g PROCESSING <100,000 ppm/kg <1,000 ppm/kg <100 ppm/kg
AGENT
The ECM portion of the wound dressing or other medical product may
be provided as a single, hydrated sheet of ECM tissue material,
such as SIS. A sheet of ECM material may be formed from one or
multiple sheets of ECM material. In multilayered ECM embodiments,
the individual sheets may be positioned in any number of
orientations relative to each other. It is further within the scope
of the disclosure for the ECM layer(s) to have any reasonable
thickness for use in the dressing or product. A sheet of ECM
material may be sized to fit the wound or other defect and to be
sufficiently flexible to conform to any complex wound/defect or
wound/defect surface. Additionally, the ECM materials may be
provided in fresh, frozen, or lyophilized (freeze-dried) forms.
Lyophilization of the ECM material may provide increased porosity
and enhanced tissue ingrowth capacity to an ECM sheet. Lyophilized
ECM materials may be used in the dried form, or they may be
hydrated prior to use.
ECM sheet materials applied to a wound or other tissue defect
surface may be fenestrated (or perforated) or meshed (or slitted)
to prevent fluid accumulation below the SIS layer. Any conventional
mechanical means for perforating or fenestrating skin grafts may be
used to fenestrate ECM materials of the present invention. The
fenestrations, or perforations, in the ECM sheet materials may
permit blood and cells from the wound or defect to migrate into the
ECM material layer(s) to start the tissue growth in the ECM
framework of the sheet material. The fenestrations may further
allow exudate materials to flow upward and become absorbed by
biocompatible substrate materials overlaying the ECM materials.
This can serve to maintain the ECM material(s) in direct contact
with the wound or defect bed, rather than floating off the bed.
Meshed ECM sheet materials are generally characterized by multiple
generally parallel rows of slits, whereby the termini of the slits
in adjacent rows are longitudinally offset from one another.
Compositions and methods for making meshed ECM sheet materials are
described in U.S. Patent Application Publication No. US
2005/0021141, the disclosures of which are expressly incorporated
by reference herein.
Additionally, ECM portions of the disclosed wound dressing or other
product may be optimally configured by stretching or by laminating
together multiple pieces, layers or strips of submucosal or other
ECM tissue compressed under e.g., dehydrating conditions in
accordance with the teachings set forth in U.S. Pat. Nos. 6,206,931
and 6,358,284, which are incorporated by reference herein. The
laminated submucosal (e.g. SIS) or other ECM assembly optionally
further physically crosslinked by partially or fully drying (down
to less than 15% moisture content) under vacuum pressure.
Alternatively, the laminated SIS or other ECM assembly is
lyophilized, instead of being vacuum dried, to increase its
porosity.
SIS in its normal sheet form has widely varying differences in its
thickness and porosity on any given piece of material. Instead of
using the SIS or other ECM material in its normally occurring sheet
form, the SIS or ECM may be cut into pieces or can be shredded or
ground into small sized bits or particles. These small pieces or
bits may then be uniformly sprayed, formed, coated or cast on any
part or substrate of the wound dressing. Malleable, hydrated pieces
of ECM material may be cast on or applied like papier mache to a
form. After the cast is dried or allowed to harden, the form can be
removed. The SIS or ECM particles can be sprayed, coated or cast
onto one or more components of the wound dressing or mandrel with
or without a binder material to enhance the physical strength of
the resulting structure.
ECM materials may be stored in a hydrated or dehydrated state.
Lyophilized or air dried submucosal or other ECM materials may be
rehydrated and used in accordance with this invention without
significant loss of its biotropic, thromboresistant or mechanical
properties.
ECM Gel Materials
ECM or submucosal tissue of the present invention may be further
processed into sheet form, chunks, or alternatively, in fluidized
or powdered forms. SIS material may be in a form of a sponge-like
or foam-like SIS (lyophilized SIS sponge, such as SURGISIS.TM.
Soft-Tissue Graft (SIS) [Cook Biotech, Inc., West Lafayette, Ind.])
capable of greatly expanding in diameter as it absorbs therapeutic
material, or non-sponge material including a sheet of SIS.
Fluidized or powdered forms of submucosa may be prepared using the
techniques described in U.S. Pat. Nos. 5,275,826 and 6,206,931, and
U.S. Pat. Appl. Publ. No. 20060201996, the disclosures of which are
expressly incorporated herein by reference in their entirety. These
forms can also be applied to other ECM materials.
Fluidized ECM materials can be advantageously applied to the wound
dressings or other products of the present invention for delivering
biofilm inhibiting and/or wound- or defect-healing promoting
agents. In one aspect, bismuth thiols and/or other
biofilm-inhibiting agents are mixed with fluidized ECM material to
form a substantially homogenous biofilm-inhibiting solution. The
fluidized ECM material may be dried or formed into a gel for direct
use.
For example, comminuted submucosal or other ECM material can be
dried by freeze drying to form a powder, which can hydrated, that
is, combined with water or buffered saline and optionally other
pharmaceutically acceptable excipients, to form a fluid ECM tissue
composition. The viscosity of fluidized ECM compositions may be
manipulated by controlling the concentration of the submucosa or
other ECM component, the degree of hydration and adjusting the pH
of the submucosal or other ECM digest. The viscosity may be
adjusted to a range of about 2 to about 300,000 cps at 25.degree.
C. Higher viscosity gel formulations can have a gel or paste
consistency and may be prepared by adjusting the pH of the digest
solutions to about 6.0 to about 7.0.
Alternatively, the fluidized ECM material may be dried and adhered
to one or more biocompatible base substrates. The resulting
construct includes an adherent biotropic fibrous mass of submucosa-
or other ECM-derived components formed on at least one substrate
surface. A dried or gelled layer of fluidized ECM may be sandwiched
or otherwise positioned between two substrate layers.
Alternatively, one or more dried or gelled layers of fluidized ECM
may be adhered to one or more base substrate layers. Preferably the
biocompatible base substrate includes or is made from ECM
materials. Any construct or wound dressing containing a layer of
fluidized ECM may be dried by lyophilization.
In a particular aspect, a submucosal or other ECM gel composition
is applied to a sheet of submucosa tissue graft material (e.g.,
OASIS.RTM. Wound Matrix, Cook Biotech Incorporated, West Lafayette,
Ind., USA). The gel composition may be prepared as in described in
U.S. Pat. No. 5,275,826, which is incorporated by reference in its
entirety herein. The gel composition may be applied to a submucosa
or other ECM tissue graft sheet material to provide a layer
thickness of about 1 to 2 mm. After the gel composition is allowed
to gel, the resulting construct may be dried by lyophilization.
The formation of the fluidized ECM components can be achieved in
any suitable manner. Any suitable source of bioremodelable ECM
material can be used to prepare a solubilized mixture including
components of the material. The liquid or flowable composition
including solubilized extracellular matrix components may be
generally prepared at follows. Briefly, the ECM material is
digested in an acidic or basic medium by contact with an
appropriate enzyme or combination of enzymes. To aid in this
digestion, the ECM material may be first reduced to a particulate
form by tearing, cutting, grinding or shearing the isolated ECM
material. Illustratively, shearing may be conducted in a fluid
medium, and grinding may be conducted with the material in a frozen
state. For example, the material can be contacted with liquid
nitrogen to freeze it for purposes of facilitating grinding into
powder form. Such techniques can involve freezing and pulverizing
submucosa under liquid nitrogen in an industrial blender.
Next, the particulate ECM material may be subjected to digestion
using any suitable enzyme in an enzymatic digestion step. Such
enzymes include for example serine proteases, aspartyl proteases,
and matrix metalloproteases. The concentration of the enzyme may be
adjusted based on the specific enzyme used, the amount of ECM to be
digested, the duration of the digestion, the temperature of the
reaction, and the desired properties of the bioremodelable fibril
mass layer forming material. In an illustrative embodiment, about
0.1% to about 0.2% of enzyme (pepsin, for example) may be used and
the digestion may be conducted under cooled conditions for a period
of time sufficient to substantially digest the ECM material. The
digestion may be conducted at any suitable temperature, preferably
at temperatures between about 4.degree. C. and about 37.degree. C.
Likewise, any suitable duration of digestion may be used, such
durations typically falling in the range of about 2 to 180 hours.
The ratio of the concentration of ECM material (hydrated) to total
enzyme usually ranges from about 25 to about 125 and more typically
the ratio is about 50, and the digestion is conducted at
approximately 4.degree. C. for approximately 24-72 hours. When an
enzyme is used to aid in the digestion, the digestion will be
performed at a pH at which the enzyme is active and more
advantageously at a pH at which the enzyme is optimally active.
Illustratively, pepsin exhibits optimal activity at pH's in the
range of about 2 to 4.
If necessary or desired, the enzymes or other disruptive agents
used to solubilize the ECM material may be removed or inactivated
before proceeding with the formation of the mass layer. Also, any
disruptive agent, particularly enzymes, that remains present and
active during storage of the tissue may potentially change the
composition and potentially the layer forming characteristics of
the solution. Enzymes, such as pepsin, may be inactivated with
protease inhibitors, a shift to neutral pH, a drop in temperature
below 0.degree. C., heat inactivation, or through the removal of
the enzyme by fractionation. A combination of these methods may be
utilized to stop digestion of the ECM material at a predetermined
endpoint, for example the ECM material may be immediately frozen
and later fractionated to limit digestion.
Illustratively, during preparation of a suitable fibrous mass layer
forming material, the ECM material may be enzymatically digested
for a sufficient time to produce a hydrolysate of ECM components.
Accordingly, the ECM may be treated with one enzyme or with a
mixture of enzymes to hydrolyze the structural components of the
material and prepare a hydrolysate having multiple hydrolyzed
components of reduced molecular weight. The length of digestion
time may be varied depending on the application, and the digestion
may be extended to completely solubilize the ECM material. In some
modes of operation, the ECM material will be treated sufficiently
to partially solubilize the material to produce a digest
composition comprising hydrolyzed ECM components and nonhydrolyzed
ECM components. The digest composition may then, in illustrative
embodiments, be further processed to remove at least some of the
nonhydrolyzed components. For example, the nonhydrolyzed components
may be separated from the hydrolyzed portions by centrifugation,
filtration, or other separation techniques known in the art.
Illustratively, preferred gel-form fibrous mass layer forming
materials may be prepared from enzymatically digested vertebrate
ECM material that has been fractionated under acidic conditions,
for example including pH ranging from about 2 to less than 7,
especially to remove low molecular weight components. Typically,
the ECM hydrolysate is fractionated by dialysis against a solution
or other aqueous medium having an acidic pH, e.g. a pH ranging from
about 2 to about 7. In addition to fractionating the hydrolysate
under acidic conditions, the ECM hydrolysate is typically
fractionated under conditions of low ionic strength with minimal
concentrations of salts such as those usually found in standard
buffers such as PBS (i.e. NaCl, KCl, Na.sub.2HPO.sub.4, or
KH.sub.2PO.sub.4) that can pass through the dialysis membrane and
into the hydrolysate. Such fractionation conditions work to reduce
the ionic strength of the ECM hydrolysate and thereby provide
enhanced gel forming characteristics.
The hydrolysate solution produced by enzymatic digestion of the ECM
material has a characteristic ratio of protein to carbohydrate. The
ratio of protein to carbohydrate in the hydrolysate is determined
by the enzyme utilized in the digestion step and by the duration of
the digestion. The ratio may be similar to or may be substantially
different from the protein to carbohydrate ratio of the undigested
ECM tissue. For example, digestion of vertebrate ECM material with
a protease such as pepsin, followed by dialysis, will form a
fractionated ECM hydrolysate having a lower protein to carbohydrate
ratio relative to the original ECM material.
Flowable ECM compositions capable of forming shape retaining gels
may be used as fibrous mass layer forming material in the present
invention. Such ECM compositions can be prepared from ECM material
that has been enzymatically digested and fractionated under acidic
conditions to form an ECM hydrolysate that has a protein to
carbohydrate ratio different than that of the original ECM
material. Such fractionation can be achieved entirely or at least
in part by dialysis. The molecular weight cut off of the ECM
components to be included in the gellable material is selected
based on the desired properties of the gel. Typically the molecular
weight cutoff of the dialysis membrane (the molecular weight above
which the membrane will prevent passage of molecules) is within in
the range of about 2000 to about 10000 Dalton, and more preferably
from about 3500 to about 5000 Dalton.
In certain forms of the ECM mass layer forming material
composition, apart from the potential removal of undigested ECM
components after the digestion step and any controlled
fractionation to remove low molecular weight components as
discussed above, the ECM hydrolysate is processed so as to avoid
any substantial further physical separation of the ECM components.
For example, when a more concentrated ECM hydrolysate material is
desired, this may be accomplished by removing water from the system
(e.g. by evaporation or lyophilization) as opposed to using
conventional "salting out"/centrifugation techniques that would
demonstrate significant selectivity in precipitating and isolating
collagen, leaving behind amounts of other desired ECM components.
Thus, in certain embodiments of the invention, solubilized ECM
components of the ECM hydrolysate remain substantially
unfractionated, or remain substantially unfractionated above a
predetermined molecular weight cutoff such as that used in the
dialysis membrane, e.g. above a given value in the range of about
2000 to 10000 Dalton, more preferably about 3500 to about 5000
Dalton.
In the manufacture of the flowable ECM material, vertebrate ECM
material may be stored frozen (e.g. at about -20 to about
-80.degree. C.) in either its solid, comminuted or enzymatically
digested forms, or the material may be stored after being
hydrolyzed and fractionated. The ECM material may be stored in
solvents that maintain the collagen in its native form and
solubility. For example, one suitable storage solvent is 0.01 M
acetic acid, however other acids may be substituted, such as 0.01 N
HCl. In one form, the fractionated ECM hydrolysate may be dried (by
lyophilization, for example) and stored in a dehydrated/lyophilized
state. The dried form may be rehydrated to prepare a flowable ECM
composition capable of forming a gel that may be used as a fibril
mass layer forming material in the present invention.
In accordance with an illustrative method of fibril mass layer
forming material preparation, the fractionated ECM hydrolysate or
other flowable ECM composition will exhibit the capacity to gel
upon adjusting the pH of a relatively more acidic aqueous medium
containing it to about 5 to about 9, more preferably about 6.6 to
about 8.0, and typically about 7.2 to about 7.8, thus inducing
fibrillogenesis and matrix gel assembly. In one embodiment, the pH
of the fractionated hydrolysate may be adjusted by the addition of
a buffer that does not leave a toxic residue, and has a
physiological ion concentration and the capacity to hold
physiological pH. Examples of suitable buffers include PBS, HEPES,
and DMEM. Illustratively, the pH of the fractionated ECM
hydrolysate may be raised by the addition of a buffered NaOH
solution to 6.6 to 8.0, more preferably 7.2 to 7.8, to facilitate
the formation of an ECM-containing gel. Any suitable concentration
of NaOH solution may be used for these purposes, for example
including about 0.05 M to about 0.5 M NaOH. In accordance with an
embodiment, the ECM hydrolysate is mixed with a buffer and
sufficient 0.25 N NaOH is added to the mixture to achieve the
desired pH.
The ionic strength of the ECM hydrolysate is believed to be
important in maintaining the fibers of collagen in a state that
allows for fibrillogenesis and matrix gel assembly upon
neutralization of the hydrolysate. Accordingly, if needed, the salt
concentration of the ECM hydrolysate material may be reduced prior
to neutralization of the hydrolysate. The neutralized hydrolysate
may be caused to gel at any suitable temperature, e.g. ranging from
about 4.degree. C. to about 40.degree. C. The temperature will
typically affect the gelling times, which may range from about 5 to
about 120 minutes at the higher gellation temperatures and about 1
to about 8 hours at the lower gellation temperatures. Typically,
the hydrolysate will be effective to self-gel at elevated
temperatures, for example at about 37.degree. C. In this regard,
preferred neutralized ECM hydrolysates will be effective to gel in
less than about ninety minutes at 37.degree. C., for example within
about 30 seconds to thirty minutes at 37.degree. C.
In alternative embodiments, additional components may be added to
the ECM hydrolysate composition before, during, or after forming
the fibrous mass layer. For example, one or more of the above
described biofilm-inhibiting agents, wound healing agents or
analgesics may be added. In certain embodiments, such materials are
added prior to formation of the fibril mass layer. This may be
accomplished for example by forming a dry mixture of a powdered ECM
hydrolysate with the additional component(s), and then
reconstituting and gelling the mixture, or by incorporating the
additional component(s) into an aqueous, ungelled composition of
the ECM hydrolysate before, during (e.g. with) or after addition of
the neutralization agent. The additional component(s) may also be
added to a formed ECM gel, e.g. by infusing or mixing the
component(s) into the gel and/or coating them onto the gel. In
certain embodiments, the gel may then be dried (e.g. by
lyophilization).
In one illustrative fibrous mass layer forming material
preparation, a particulate ECM material may be added to an ECM
hydrolysate composition, which may then be incorporated in a formed
gel and ultimately in a dried mass. Such particulate ECM materials
may be prepared by cutting, tearing, grinding or otherwise
comminuting an ECM starting material. For example, a particulate
ECM material having an average particle size of about 50 microns to
about 500 microns may be included in the gellable ECM hydrolysate,
more preferably about 100 microns to about 400 microns. The ECM
particulate may be added in any suitable amount relative to the
hydrolysate, with preferred ECM particulate to ECM hydrolysate
weight ratios (based on dry solids) being about 0.1:1 to about
200:1, more preferably in the range of about 1:1 to about 100:1.
The inclusion of such ECM particulates in the ultimate gel or
fibril mass layer forming material may serve to provide additional
material that may function to provide bioactivity to the gel (e.g.
itself including FGF-2 and/or other growth factors or bioactive
substances as discussed herein) and/or serve as scaffolding
material for tissue ingrowth.
In certain embodiments, flowable ECM compositions to be used as
fibrous mass layer forming material in the invention may be
disinfected by contacting an aqueous medium including ECM
hydrolysate components with an oxidizing disinfectant. This mode of
disinfection provides an improved ability to recover a disinfected
ECM hydrolysate that exhibits the capacity to form beneficial gels.
In certain preparative methods, an aqueous medium containing ECM
hydrolysate components may be disinfected by providing a peroxy
disinfectant in the aqueous medium. This can be advantageously
achieved using dialysis to deliver the peroxy disinfectant into
and/or to remove the peroxy disinfectant from the aqueous medium
containing the hydrolysate. In certain disinfection techniques, an
aqueous medium containing the ECM hydrolysate is dialyzed against
an aqueous medium containing the peroxy disinfectant to deliver the
disinfectant into contact with the ECM hydrolysate, and then is
dialyzed against an appropriate aqueous medium (e.g. an acidic
aqueous medium) to at least substantially remove the peroxy
disinfectant from the ECM hydrolysate. During this dialysis step,
the peroxy compound passes through the dialysis membrane and into
the ECM hydrolysate, and contacts ECM components for a sufficient
period of time to disinfect the ECM components of the hydrolysate.
In this regard, typical contact times will range from about 0.5
hours to about 8 hours and more typically from about 1 hour to
about 4 hours. The period of contact will be sufficient to
substantially disinfect the digest, including the removal of
endotoxins and inactivation of virus material present. The removal
of the peroxy disinfectant by dialysis may likewise be conducted
over any suitable period of time, for example over a duration of
about 4 to about 180 hours, more typically of about 24 to about 96
hours. In general, the disinfection step will desirably result in a
disinfected ECM hydrolysate composition having sufficiently low
levels of endotoxins, viral burdens, and other contaminant
materials to render it suitable for use as a fibril mass layer
forming material. Endotoxin levels below about 2 endotoxin units
(EUs) per gram (dry weight) are preferred, more preferably below
about 1 EU per gram, as are virus levels below 100 plaque forming
units per gram (dry weight), more preferably below 1 plaque forming
unit per gram.
The aqueous ECM hydrolysate composition may be a substantially
homogeneous solution during the dialysis step for delivering the
oxidizing disinfectant to the hydrolysate composition and/or during
the dialysis step for removing the oxidizing disinfectant from the
hydrolysate composition.
Alternatively, the aqueous hydrolysate composition may include
suspended ECM hydrolysate particles, optionally in combination with
some dissolved ECM hydrolysate components, during either or both of
the oxidizing disinfectant delivery and removal steps. Dialysis
processes in which at least some of the ECM hydrolysate components
are dissolved during the disinfectant delivery and/or removal steps
are preferred and those in which substantially all of the ECM
hydrolysate components are dissolved are more preferred.
The disinfection step may be conducted at any suitable temperature,
and will typically be conducted between about 0.degree. C. and
about 37.degree. C., more typically between about 4.degree. C. and
about 15.degree. C. During this step, the concentration of the ECM
hydrolysate solids in the aqueous medium may range between about 2
mg/ml and about 200 mg/ml, and may vary somewhat through the course
of the dialysis due to the migration of water through the membrane.
In certain embodiments, a relatively unconcentrated digest is used,
having a starting ECM solids level of about 5 mg/ml to about 15
mg/ml. In other embodiments, a relatively concentrated ECM
hydrolysate is used at the start of the disinfection step, for
example having a concentration of at least about 20 mg/ml and up to
about 200 mg/ml, more preferably at least about 100 mg/ml and up to
about 200 mg/ml. It has been found that the use of concentrated ECM
hydrolysates during this disinfection processing results in an
ultimate gel composition having higher gel strength than that
obtained using similar processing with a lower concentration ECM
hydrolysate. Accordingly, processes which involve the removal of
amounts of water from the ECM hydrolysate resulting from the
digestion prior to the disinfection processing step are preferred.
For example, such processes may include removing only a portion of
the water (e.g. about 10% to about 98% by weight of the water
present) prior to the dialysis/disinfection step, or may include
rendering the digest to a solid by drying the material by
lyophilization or otherwise, reconstituting the dried material in
an aqueous medium, and then treating that aqueous medium with the
dialysis/disinfection step.
In an illustrative fibrous mass layer forming material preparation
embodiment, the disinfection of the aqueous medium containing the
ECM hydrolysate may include adding the peroxy compound or other
oxidizing disinfectant directly to the ECM hydrolysate, for example
being included in an aqueous medium used to reconstitute a dried
ECM hydrolysate or being added directly to an aqueous ECM
hydrolysate composition. The disinfectant may then be allowed to
contact the ECM hydrolysate for a sufficient period of time under
suitable conditions (e.g. as described above) to disinfect the
hydrolysate, and then removed from contact with the hydrolysate. In
one embodiment, the oxidizing disinfectant may then be removed
using a dialysis procedure as discussed above. In other
embodiments, the disinfectant may be partially or completely
removed using other techniques such as chromatographic or ion
exchange techniques, or may be partially or completely decomposed
to physiologically acceptable components. For example, when using
an oxidizing disinfectant containing hydrogen peroxide (e.g.
hydrogen peroxide alone or a peracid such as peracetic acid),
hydrogen peroxide may be allowed or caused to decompose to water
and oxygen, for example in some embodiments including the use of
agents that promote the decomposition such as thermal energy or
ionizing radiation, e.g. ultraviolet radiation.
In an alternative fibrous mass layer forming material preparation,
the oxidizing disinfectant may be delivered into the aqueous medium
containing the ECM hydrolysate by dialysis and processed
sufficiently to disinfect the hydrolysate (e.g. as described
above), and then removed using other techniques such as
chromatographic or ion exchange techniques in whole or in part, or
allowed or caused to decompose in whole or in part as discussed
immediately above.
Peroxygen compounds that may be used in the disinfection step
include, for example, hydrogen peroxide, organic peroxy compounds,
and preferably peracids. Such disinfecting agents are used in a
liquid medium, preferably a solution, having a pH of about 1.5 to
about 10.0, more desirably of about 2.0 to about 6.0. As to peracid
compounds that may be used, these include peracetic acid,
perpropioic acid, and/or perbenzoic acid. Peracetic acid is the
most preferred disinfecting agent for purposes of the present
invention.
When used, peracetic acid is desirably diluted into about a 2% to
about 50% by volume of alcohol solution, preferably ethanol. The
concentration of the peracetic acid may range, for instance, from
about 0.05% by volume to about 1.0% by volume. Most preferably, the
concentration of the peracetic acid is from about 0.1% to about
0.3% by volume. When hydrogen peroxide is used, the concentration
may range from about 0.05% to about 30% by volume. More desirably
the hydrogen peroxide concentration is from about 1% to about 10%
by volume, and most preferably from about 2% to about 5% by volume.
The solution may or may not be buffered to a pH from about 5 to
about 9, with more preferred pH's being from about 6 to about 7.5.
These concentrations of hydrogen peroxide may be diluted in water
or in an aqueous solution of about 2% to about 50% by volume of
alcohol, most preferably ethanol. For additional information
concerning preferred peroxy disinfecting agents useful in certain
disinfecting embodiments of the present invention, reference may be
made, for example, to U.S. Pat. No. 6,206,931.
In certain embodiments, flowable, ECM-based fibrous mass layer
forming materials of the present invention may be prepared to have
desirable properties for manufacturing, handling and use. For
example, fluidized ECM hydrolysates may be prepared in an aqueous
medium, which can provide a fibril mass layer of forming material.
Such prepared aqueous mediums can have any suitable level of ECM
hydrolysate therein. Typically, the ECM hydrolysate will be present
in the aqueous medium at a concentration of about 1 mg/ml to about
200 mg/ml, more typically about 2 to about 120 mg/ml. Furthermore,
flowable ECM compositions can be prepared so that in addition to
neutralization, heating to physiologic temperatures (such as
37.degree. C.) will substantially reduce the gelling time of the
material.
In the formation of medical graft materials of the invention, the
liquid or otherwise flowable composition containing solubilized ECM
components may be applied to the substrate material in any suitable
fashion. For example, an amount of an ECM or other base sheet
material may be spread, potentially within a mold, cast, or other
structure for retaining and/or shaping the liquid composition to be
applied. The flowable composition may then be added to the surface
of the ECM or other base sheet material to a desired thickness or
depth. In certain embodiments as discussed above, the flowable
composition will be capable of forming a gel. This gel or other
liquid-containing composition may then be dried to form a dried
cake or mass that includes fibrous collagen derived from the ECM
material, desirably along with one or more bioactive components
native to the ECM material, as discussed above.
Bismuth Thiols
Bismuth thiols of the present invention are designed to promote
wound healing by preventing, reducing or eliminating biofilm
development in the wound. Bismuth thiols include a group of
biocidal agents having potent, broad spectrum antibacterial
activity (Domenico et al., Antimicrob. Agents Chemother., vol. 41,
pp. 1697-1703 (1997); and Domenico et al., Antimicrob. Agents
Chemother., vol. 45, pp. 1417-1421 (2001). Bismuth thiols as used
herein may also exhibit certain antifungal properties as well.
Exemplary bismuth thiols include bismuth-1,2-ethanedithiol,
bismuth-2-mercaptoethanol, bismuth-3,4-dimercaptotoluene,
bismuth-pyrithione, bismuth-2,3-dimercaptopropanol,
bismuth-1,3-propanedithiol, bismuth-dithiothreitol,
bismuth-3-mercapto-2-butanol. Additional bismuth thiols are
described in U.S. Pat. No. 6,086,921, which is incorporated by
reference herein. Preferably, the bismuth thiol is a bismuth
dithiol. In a particularly preferred embodiment, the bismuth thiol
is bismuth-1,2-ethanedithiol.
Bismuth thiols are incorporated into the bioremodelable material in
an amount sufficient to prevent or reduce biofilm formation,
prevent or reduce biofilm growth, or to remove or disrupt an
existing bacterial biofilm. Bismuth thiol amounts may be varied
depending on the efficacy of the agent in accordance with, for
example, U.S. Pat. No. 6,380,248 and Domenico et al., Antimicrob.
Agents Chemother., Vol. 45, No. 5, pp. 1417-1421, 2001.
Other Biofilm-Inhibiting Agents
In accordance with the present invention, the wound dressing or
other medical product may further comprise at least other
biofilm-inhibiting agent. The other biofilm-inhibiting agents
include iron-sequestering glycoproteins, such as lactoferrin,
ovotransferrin, and serrotransferrin; xylitol; chelating agents,
such as EDTA, EGTA, and DTPA; biocidal agents, antibiotics, quorum
sensing inhibitors, and surfactants. The other biofilm-inhibiting
agent(s) may be administered with the bismuth thiol or may be
administered separately. Generally, these biofilm-inhibiting agents
will have minimal adverse effects on host cells in e.g., a wound
bed area. Among the other biofilm-inhibiting agents, lactoferrin,
xylitol, and/or antibiotics are preferred agents for use in the
present invention.
The use multiple biofilm-inhibiting agents acting though multiply
distinct mechanisms affecting biofilm formation and maintenance can
provide synergistic effects with regard to biofilm management and
wound healing. For example, biofilm-inhibiting agents can be
selected to include antibiotics or biocides killing microbes, as
well as agents affecting attachment to wound bed surfaces,
cell-cell communication (e.g., quorum sensing) between
microorganisms in the biofilm, and/or biofilm structural
organization.
Lactoferrin is naturally present in human secretions (saliva,
tears, mucous, milk) and constitutes an important part of a host's
natural immunity to microorganisms. In addition to sequestering
iron, an essential bacterial nutrient, lactoferrin also acts as a
serine protease inducing degradation of bacterial secreted proteins
necessary for attachment. Moreover, lactoferrin has bactericidal
activity reflected in its ability to bind to gram-negative
bacterial lipopolysaccharide (LPS) portions, increasing their outer
membrane permeability, and leading to bacterial cell death.
Lactoferrin is also known to enhance the bacterial cell killing
ability of activated neutrophils.
Xylitol is a sugar alcohol naturally occurring in certain plants,
has been shown to be active in blocking adhesion of pathogenic
gram-positive bacteria to host surfaces and in treating
biofilm-based diseases of skin and dental plaque.
Representative biocidal agents for use in the present invention
include iodine compounds and derivatives thereof, such as cadexomer
iodine and povidone iodine (PVP-I); silver compounds and
derivatives thereof, such as silver sulfadiazine (brand names:
Silvadene, SSD, SSD AF1 Thermazene) and silver nitrate
(AgNO.sub.3); divalent metal chelating agents, such as EDTA and
EGTA; salicylic acid; chlorine dioxide; isothiazolone, derivatives
thereof, compounds having isothiazolone functions, 3-isothiazolone,
5-chloro-2-methyl-3-isothiazolone,
1-methyl-3,5,7-triaza-1-azoniatricyclo(3.3.1.1) deoane chloride,
4,5-dichloro-2-octyl-3 isothiazolone, 2-bromo-2-nitropropanediol,
5-bromo-5-nitro dioxane, thiocyanomethylthiobenzothiazole,
4,5-dichloro-2-octyl-3-isothiazolone and 2n-octyl-3-isothiazolone,
tetrachloroisophalonitrile, 1,2-benzisothiazolin-3-one,
2-methyl-4,5-trimethylene-4-isothiazolin-3-one, 5-chloro-2-methyl-4
isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one,
4-(2-nitrobutyl)morpholine, beta-nitrostyrene ("NS"),
beta-bromo-beta-nitrostyrene ("BNS"), methylehloro/isothiazolone
("IZN"), methylenebisthiocyanate ("MBT"),
2,2dibrortmo-3-nitrilopropionamide ("DBNPA"),
2-bromo-2-brornomethyl-glutaronitrile ("BBMGN"),
alkyldimethylbenzylammoniutn chloride ("ADBAC"), and
beta-tiitrovinyl furan ("NVF"), 2-methyl-3-isothiazolone, methylene
bisthiocyanate, p-tolyidiiodotnethyl sulfone,
2-methylthio-4-tertbutylamino-6-cyclopropyl-amino-s-tiiazine,
N,N-dimethyl-N'-phenyl-(N'fluorodiehloromethylthio)sulfa-inide,
antibiotics, sulfamides, tetracycline, isothiazolone derivatives,
N-(cyclo)alkyl-isothiazolone, benzisothiazolin-3-one, and mixtures
of the foregoing.
Other examples of biocidal agents that may be combined with one or
more of biofilm-inhibiting compounds listed above include bicyclic
oxazolidoines and their mixtures, amine-based bactericide,
polyacrolein copolymer, 4,4-dimethyloxazolidine,
2((hydroxymethyl)-amino) ethanol, mixtures of
1,2-benzisothiazolone-3-one with one or more amines,
tetrahydro-3,5-dimethyl-2H-1,3,5-thiadiazitie-2-thione,
1,2-benzisothiazolin-3-one, tetrachloroisophthalonitrile,
N-cyclopropyl-N-(1,1-dimethylethyl)-6-(methylthio)-1,3;
5-triazine-2,4-diamine, mixtures of
N-cyclopropyl-N-(1,1-dimethylethyl)-6-(methylthio)-1,3,5-triazine-2,4-dia-
mine with tetrachloroisophthalonitrile-, mixtures of
tetrachloroisophthalonitrile with 3-iodo-2-propynylbutyl carbamate,
N-(trichloromethylthio)-phthalimide, 3iodo-2-propynylbutyl
carbamate, tetrachloroisophthalonitrile, and mixtures of the
foregoing, which are described in U.S. Application Publication No.
2006/0014285.
Non-limiting examples of antibiotics that may be used in connection
with the present invention include aminopenicillins (penicillin,
amoxicillin, and their congeners); cephalosporins; cycloserine;
macrolides (erythromycin, clarithromycin, azithromycin,
roxithromycin); quinolones; sulfonamides; rifamycins, including
rifampin (RIFADIN; RIMACTANE); thienamycins (imipenem);
tetracyclines (chlortetracycline, oxytetracycline, demeclocycline,
methacycline, doxycycline, and minocycline); cefaclor, cefuroxime,
cefprozil, chloramphenicol, ciprofloxacin, clindamycin, ethabuto,
dicloxacillin, erythromycin, metronidazole, ofloxacin,
griseofulvin, sulfisoxazole, griseofulvin, cephalexin, terbinafine,
levofloxacin, loracarbef, nitrofurantoin, minocycline, polymyxin,
vancomycin, tobramycin, clotrimazole, nystatin, ketoconazole,
cefdinir, ampicillin, trimethoprim-sulfamethoxazole, itraconazole,
cefixime, mebendazole, doxycycline, sparfloxacin, azithromycin,
including analogs, and mixtures of the foregoing. Antibiotics are
preferably selected to target microorganisms native to the wound
bed. Preferably microbial target of the antibiotic is sufficiently
different from its physiological counterpart to preclude adverse
effects in the host cells of a subject in need of wound healing
treatment.
Non-limiting examples of anti-fungal antibiotic agents include
amphotericin B, flucytozine, imidazoles and triazoles,
ketoconazole, itraconazole, fluconazole, ciclopirox olamine,
haloprogin, tolnaftate, naftifine, terbinafine, and polyene
antifungal antibiotics (nystatin).
Non-limiting examples of antiviral agents include antiretroviral
agents (didanosine, stavudine, zalcidabine, zidovudine),
antiherpesvirus agents (acyclovir, famciclovir, foscarnet,
trifluridine, vidarabile), and other antiviral agents (amantadine,
interferon alpha, ribavirin, rimantadine).
Non-limiting examples of quorum sensing antagonists include
N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL),
N-butyrl-L-homoserine lactone (BHL), and analogs thereof.
Surfactants may be used in conjunction with other
biofilm-inhibiting substances to disrupt biofilms. Previous studies
have mutants defective in quorum sensing lose the ability to resist
biofilm disruption in the presence of 0.2% sodium dodecyl sulfate
(SDS) treatment. Non-limiting examples of surfactants include SDS;
amidoalkyl betaines of fatty acids, including undecylene amidoalkyl
betaine, cocamidoalkyl betaine, lauramidoalkyl betaine, and
ricinolamidoalkyl betaine; Tween.RTM. 80, cyclic lipopeptide,
cyclic heptapeptide, surfactin, and serrawettin.
The biofilm-inhibiting compounds or compositions of the present
invention may be administered in connection with pharmaceutically
acceptable carriers known to those of skill in the art.
Biofilm-inhibiting compounds or compositions may be coupled with
soluble polymers as targetable carriers. This may provide
controlled release of agents that may be otherwise toxic to host
cells. Such polymers may include polyvinylpyrrolidone, pyran
copolymer, polyhydroxypropylmethacrylamide phenyl,
polyhydroxyethylaspartamide-phenol, or polyethyleneoxide-polylysine
substituted with palmitoyl residues. Other biodegradable polymers
useful for coupling and controlled release of a biofilm-inhibiting
agents include polylactic acid, polyglycolic acid, copolymers of
polylactic and polyglycolic acid, polyepsilon caprolactone,
polyhydroxy butyric acid, polyorthoesters, polyacetals,
polydihydropyrans, polycyanoacrylates and cross-linked or
amphipathic block copolymers of hydrogels.
Bismuth thiols and other biofilm-inhibiting agents may be
formulated to provide controlled release over time, for example,
days, weeks, months or years, as the ECM is degraded or eroded. In
an exemplary embodiment, degradation of the ECM is modulated by an
agent that decreases (e.g., via a peptide, protein, or chemical
protease, such as, for example, aprotinin) or increases (e.g., a
protease) the rate of degradation and/or erosion of the ECM.
Alternatively, the bismuth thiols and other biofilm-inhibiting
agents may comprise a microsphere composition which is attached to
or incorporated within the ECM. In this embodiment, the ECM need
not degrade in order to produce a time released effect of the
bismuth thiols and other biofilm-inhibiting agents. Release
properties can also be determined by the size and physical
characteristics of the microspheres.
Bismuth thiols and other biofilm-inhibiting agents may also
include, for example, adjuvants and additives, such as stabilizers,
fillers, antioxidants, catalysts, plasticizers, pigments, and
lubricants, to the extent such ingredients do not diminish the
utility of the biofilm-inhibiting agent for its intended
purpose.
Other Bioactive Agents
The wound healing process is regulated by the integrated actions of
growth factors, cytokines, proteases, and extracellular matrix
components (see e.g., Lobmann et al., Diabetes Care, vol. 28, no.
2, February, 2005). Chronic wounds exhibit various deficiencies and
imbalances in key proteases, cytokines and growth factors. For
example, poorly healing chronic wounds are typically characterized
by excessive inflammation, a prolonged period of inflammation, and
excessive protease activity, possibly the result of wound bed
biofilms. Bacterial endotoxins, fragments of extracellular matrix,
and cell detritus maintain this inflammation, which is evidenced by
a large number of neutrophil granulocytes in the wound secreting
various proinflammatory cytokines, particularly tumor necrosis
factor-.alpha. (TNF-.alpha.) and interleukin-1.beta. (IL-1.beta.),
which can contribute to a persistent inflammatory status. The
prolonged inflammatory status contributes to the elevated protease
levels, which can lead to degradation of matrix proteins and growth
factors essential for healing. Indeed, as compared to acute wounds,
chronic wounds typically exhibit reduced levels of growth factors,
such as PDGF, b-FGF, EGF, and TGF-.beta., and protease regulators,
including tissue inhibitors of metalloproteinases (TIMPs), such as
TIMP-1. The imbalanced integration of growth factors, cytokines,
proteases, and extracellular matrix components in chronic wounds
can ultimately result in a failure of the wound to heal.
Accordingly, wound dressings of the present invention may further
include wound healing agents and protease inhibitors as
appropriate. Choice of wound healing factors may depend on whether
the wound is acute or chronic.
Exemplary wound healing agents include growth factors, cytokines,
and protease inhibitors. Exemplary growth factors include
TGF-related growth factors (TGF-.beta.1, TGF-.beta.2, TGF-.beta.3);
PDGF-related growth factors (PDGF-M, PDGF-BB, VEGF); FGF-related
growth factors (a-FGF, b-FGF, KGF); IGF-related growth factors
(IGF-1, IGF-II, insulin); EGF-related growth factors (EGF, HB-EGF,
TGF-.alpha., amphiregulin, betacellulin); CTGF; and combinations,
analogs and/or recombinant derivatives thereof. Exemplary cytokines
include proinflammatory cytokines, such as TNF-.alpha., IL-1, IL-2,
IL-6, IL-8, .gamma.-interferon; anti-inflammatory cytokines, such
as IL-4 and IL-10; and combinations, analogs and/or recombinant
derivatives thereof.
Medical products of the present invention may also include ECM
sheet materials formulated to include exogenous fibronectin, as
well as exogenous heparin or exogenous heparin sulfate bound
thereto, and/or exogenous heparin- and heparin sulfate-binding
growth factors bound to the fibronectin-bound exogenous heparin-
and heparin-sulfates. Methods of making and using
fibronectin-modified ECM materials are described in U.S.
Provisional Application No. 60/618,965, filed Oct. 15, 2004, and in
International Application Number PCT/US2005/036773, filed Oct. 14,
2005, the disclosure of which are expressly incorporated by
reference herein.
Exemplary protease inhibitors include matrix metalloproteinase
(MMP) inhibitors, including but not limited to TIMP-1, TIMP-2, and
TIMP-3; and doxycycline, which has been shown to reduce
inflammation and improve healing of chronic diabetic foot ulcers
treated with a topical doxycycline gel (Lobmann et al., Diabetes
Care, vol. 28, no. 2, February, 2005). MMP substrates, such as
gelatin, may also be applied to the dressings of the present
invention. Recent studies have shown the use of gelatin in wound
dressings to reduce MMP activity and to improve healing in chronic
wounds (Lobmann et al., Diabetes Care, vol. 28, no. 2, February,
2005).
Inasmuch as wounds require effective pain management, analgesics
may be incorporated into the wound dressings or applied to wounds
in conjunction with the wound dressings. Analgesic agents may be
used for pain relief or pain suppression, especially for treatment
of burns. Examples of the analgesic agents include, but are not
limited to, previously mentioned nonsteroidal anti-inflammatory
drugs, and opioids, such as morphine, methadone, codeine,
etorphine, naloxone, and others.
Incorporation of Bioactive Agents into Bioremodelable Materials
Bismuth thiols and other biofilm-inhibiting agents, wound healing
agents, and/or analgesic agents may be carried by bioremodelable
sheet materials in any suitable fashion. For example, the bioactive
agents may be exogenously incorporated into the carrier during
their preparation or covalently attached to the carrier when
preparing the wound dressing. Alternatively, bioactive agents may
be added to the carrier after preparation of the carrier, for
example, by impregnating (e.g., dry powder coating, soaking,
coating, spraying, painting) or otherwise applying the bioactive
agent(s) to the carrier using methods known to those of skill in
the art.
Additionally, the exogenous bioactive agents may be applied in the
form of a liquid medium containing the agent(s), such as a solution
or suspension, which is contacted with all or only one or more
portions of the sheet, after which the sheet can be dried to leave
the agent(s) in place. Contact between the liquid medium and the
sheet can be achieved in any suitable manner, including for example
immersion, spraying, coating or otherwise. After drying, the drug
may be substantially homogenously dispersed through the sheet, or
may be selectively applied to regions of the sheet. Alternatively,
the drug can be a powder which is applied, by spraying, rubbing or
otherwise coating, to one or both sides of the sheet. Methods for
applying bioactive agents to one or more ECM layers in monolayer or
multi-layer sheet constructs are disclosed in U.S. Patent
Application Publication No. US 2006/0251702, the disclosures of
which are expressly incorporated by reference herein.
In another aspect, the bismuth thiols and/or other
biofilm-inhibiting or wound healing agents are mixed with fluidized
ECM- or other bioremodelable materials to form a substantially
homogenous biofilm-inhibiting wound dressing solution. The
fluidized material may be dried or formed into a gel for direct
use.
Methods for Treating Wounds or Other Tissue Defects
The above-described wound dressings, graft materials or medical
products may be used to treat a variety of wounds or tissue
defects, including partial and full thickness wounds, diabetic
ulcers, venous ulcers, chronic vascular ulcers, leg ulcers,
pressure ulcers, decubitus, ulcus cruris, tunneled/undermined
wounds, fistulae, surgical wounds (such as donor site wounds for
autografts, post-Moh's surgery wounds, post-laser surgery wounds,
wound dehiscence), hernias, trauma wounds (such as abrasions,
lacerations, burns, and skin tears), draining wounds, and the like.
In a preferred embodiment, a wound dressing in accordance with the
present invention is used to treat a chronic wound. In other
preferred embodiments, a wound dressing in accordance with the
present invention is used to treat a hernia or a fistula.
The bismuth thiols and/or other biofilm-inhibiting agents may be
incorporated into or onto a suitable bioremodelable source material
before application to a wound or defect or may be independently
applied to a wound/defect in parallel with application of the
bioremodelable material. The wound dressings described herein may
be utilized in conjunction with conventional wound management
practices, including repeated dressing exchanges and debridement of
the wound area.
For example, when preparing the wound for treatment in a
conventional fashion, the physician, veterinarian or other user of
the wound dressing materials of the invention may, for example,
include cleaning and/or debridement of the wound with water,
physiologic saline or other solutions, and potentially also
treating the wound with antibiotics or other therapeutic agents.
Methods for treating wounds with bioremodelable sheet constructs
can be found in the manufacturer's instructions for OASIS.RTM.
Wound Matrix (Cook Incorporated, West Lafayette, Ind.).
Bioremodelable sheet constructs and other dressing materials can
applied to the wound in a fashion to facilitate and promote healing
of the wound. In this regard, the inventive sheet constructs, for
example, may be applied in a dehydrated, partially hydrated, or
fully hydrated state. Once applied to a wound, the modified sheet
construct will hydrate (if not previously hydrated) and remain
generally in place either alone or in combination with other wound
dressing materials applied below or on top of the modified ECM
material.
Those skilled in the art of treating damaged or diseased tissue in
humans will know or can determine optimal dosages of the
biofilm-inhibiting agents for incorporation into the wound dressing
to treat a subject in need. In general, the effective therapeutic
amount is adjusted for body surface area requiring such treatment.
A therapeutically effective amount of a biofilm-inhibiting agent in
the wound dressing is expected to vary from about 0.1 milligram per
kilogram of body weight per day (mg/kg/day) to about 100 mg/kg/day.
Dosage determinations of biofilm-inhibiting agents may be made by a
physician, veterinarian, or attending clinician. The dosages may be
varied depending upon the requirements of the patient, the severity
of the condition being treated and the particular agent being
employed. In determining the dose, a number of factors are
considered by the attending clinician, including, but not limited
to: the specific tissue to be treated; pharmacodynamic
characteristics of the particular agent; the desired time course of
treatment; the species of mammal; its size, age, and general
health; the specific disease involved; the degree of or involvement
or the severity of the disease; the response of the individual
patient; the particular compound administered; the mode of
administration; the bioavailability characteristics of the
preparation administered; the dose regimen selected; the kind of
concurrent treatment; and other relevant circumstances.
By exogenously incorporating bismuth thiols and other
biofilm-inhibiting, and/or wound healing agents into the medical
products of the present invention, increased biofilm inhibition,
reduction, or removal can be achieved compared to conventional
medical products. In addition, incorporating these agents may
further enhance removal of biofilms by debridement and may reduce
the necessity for repeated debridement.
For the purpose of promoting a further understanding of embodiments
of the invention and their features and advantages, the following
specific Examples are provided. It will be understood that these
Examples are illustrative, and not limiting, in nature.
EXAMPLE 1
This Example was performed to test the antimicrobial activity of a
sample of small intestinal submucosa coated with a bismuth thiol
preparation.
Seven bismuth thiol compounds (50 mg each) were obtained from
Microbion, Inc. (Bozeman, Mont.). These compounds included 2
preparations of bismuth-1,2-ethanedithiol (BisEDT) (Molar ratio
1:1.5), 2 preparations of bismuth-2,3-dimercaptopropanol (BisBAL)
(1:1 and 1:1.5), bismuth dithioethylhritol (BisERY) (1:1.5),
bismuth-3,4-dimercatotoluene (BisTOL) (1:1.5) and bismuth
butanedithiol (BisBDT) (1:1.5). Purified small intestinal submucosa
(SIS) (Cook Biotech Incorporated, West Lafayette, Ind.) was made
into wetted 4-layer constructs which were laminated together by
lyophilization and subsequently cut into 15 mm discs (.about.1.76
cm.sup.2) using a disc punch. Bismuth thiols were resuspended in
dimethyl sulfoxide (DMSO) and diluted to appropriate concentrations
for coating. Discs were coated with the bismuth thiol solutions at
three final concentrations using 17.6 .mu.l of the appropriate
solution per disc for a final concentration of 1, 10, or 100
.mu.g/cm.sup.2. The discs were subsequently placed in a lyophilizer
overnight to remove the DMSO solvent. Control discs were prepared
using 17.6 .mu.l of DMSO without a bismuth thiol preparation. 3
bacteria were used for this experiment: S. aureus (ATCC #25983), P.
aeruginosa (ATCC #27853), and E. coli (ATCC #25922). Bacteria was
grown in Tryptic Soy Broth for 16-24 hours before placing a lawn on
Tryptic Soy Agar in 100 mm Petri dishes.
Sixty six samples were tested in total (7 bismuth thiol
preparations 3 doses=21+1 control=22.times.3 bacteria=66). A lawn
of one of the three bacteria investigated was swabbed onto the
Tryptic Soy Agar. The bismuth thiol coated disc was placed
approximately in the center of the plate and rehydrated with
.about.50 .mu.l of sterile saline. Plates were incubated inverted
for 24 hours at which time pictures were taken with a digital
camera. Zones-of-inhibition were categorized by the approximate
distance of bacterial growth inhibition from the disc edge. The
effectiveness was ranked as follows: (-) for no growth retardation,
(+) for growth retardation at <1 mm from disc edge, (++) for
growth retardation 1-3 mm from disc edge, and (+++) for growth
retardation greater than 3 mm from disc edge. Results are shown
below in Tables 2-4.
TABLE-US-00002 TABLE 2 (S. aureus) Conc. Bis Bis Bis Bis Bis Bis
Bis (.mu.g/cm.sup.2) EDT1 EDT2 BAL1 BAL2 ERY TOL BDT 1 + ++ + + - -
- 10 ++ ++ +++ +++ ++ + ++ 100 +++ +++ +++ +++ +++ + +++
TABLE-US-00003 TABLE 3 (P. aeruginosa) Conc. Bis Bis Bis Bis Bis
Bis Bis (.mu.g/cm.sup.2) EDT1 EDT2 BAL1 BAL2 ERY TOL BDT 1 - - - -
- - - 10 + - + + ++ - + 100 + ++ +++ +++ +++ - ++
TABLE-US-00004 TABLE 4 (E. coli) Conc. Bis Bis Bis Bis Bis Bis Bis
(.mu.g/cm.sup.2) EDT1 EDT2 BAL1 BAL2 ERY TOL BDT 1 - - - - - - - 10
- + ++ ++ ++ - ++ 100 + + +++ +++ +++ - ++
There was no zone-of-inhibition detected for the control discs. As
the Tables indicate, each plate containing a disc coated with a
bismuth thiol illustrated some antimicrobial activity. This
suggests that bismuth thiols can be used in conjunction with a
medical device to effectively retard bacterial growth on, and in
the vicinity of, the medical device.
EXAMPLE 2
This Example was performed to further test the antimicrobial
activity of BisERY, BisEDT, and BisBAL when coated onto a sample of
small intestinal submucosa (SIS).
BisEDT (Molar ratio 1:1.5), BisBAL (1:1.5), and BisERY (1:1.5) (50
mg each) were obtained from Microbion, Inc. Small intestinal
submucosa (SIS) was obtained and processed into 4-layer lyophilized
sheets as described in Example 1. The sheets were subsequently cut
into 15 mm discs (.about.1.76 cm.sup.2) using a disc punch. Bismuth
thiols were resuspended in dimethyl sulfoxide (DMSO) and diluted to
0.35 .mu.g/.mu.l for coating. Discs were coated with the bismuth
thiol solutions 50 .mu.l of the appropriate solution per disc for a
final concentration 10 .mu.g/cm.sup.2 (0.35 .mu.g/.mu.l*50
.mu.l=17.5 .mu.g/1.76 cm.sup.2=10 .mu.g/cm.sup.2). The discs were
subsequently placed in a lyophilizer overnight to remove the DMSO
solvent. Control discs were prepared using 50 .mu.l of DMSO without
a bismuth thiol preparation. 3 bacteria were used for this
experiment: S. aureus (ATCC #25983), P. aeruginosa (ATCC #27853),
and E. coli (ATCC #25922). Bacteria was grown in Tryptic Soy Broth
for 16-24 hours before placing a lawn on Tryptic Soy Agar in 100 mm
Petri dishes.
Thirty samples were tested in total (3 bismuth thiol preparations @
n=3.fwdarw.9+1 control=10.times.3 bacteria=30). A lawn of one of
the three bacteria investigated was swabbed onto the Tryptic Soy
Agar. The bismuth thiol coated disc was rehydrated for .about.5
minutes in sterile saline and placed approximately in the center of
the plate. Plates were incubated inverted for 24 hours at which
time pictures were taken with a digital camera. Zones-of-inhibition
were measured using image analysis software (Spot RT) by
determining the distance of bacterial growth inhibition from the
disc edge. Results are shown below in Table 5.
TABLE-US-00005 TABLE 5 Bacterial BisEDT BisBAL BisERY S. aureus
6.83 7.27 0.80 P. Aeruginosa 2.06 0.16 0.00 E. Coli 1.28 1.50
0.00
There was no zone-of-inhibition detected for the control discs. As
indicated in Table 5, BisERY coated discs showed a small
zone-of-inhibition for S. aureus and no effect for P. aeruginosa or
E. coli. BisEDT and BisBAL worked similarly for S. aureus and E.
coli with BisBAL showing slightly greater zones-of-inhibition.
BisEDT was more effective against P. aeruginosa than BisBAL.
This Example further demonstrates the ability of an ECM material
coated with BisERY, BisEDT or BisBAL to retard bacterial growth and
suggests that such compounds can be used in conjunction with a
medical device for this purpose.
EXAMPLE 3
This Example was performed to test the effects of various
rehydration times of an SIS disc coated with either BisEDT or
BisBAL.
BisEDT (Molar ratio 1:1.5) and BisBAL (1:1.5) (50 mg each) were
obtained from Microbion, Inc. Small intestinal submucosa (SIS) was
obtained and processed into 4-layer lyophilized sheets as described
in Example 1. The sheets were subsequently cut into 15 mm discs
(.about.1.76 cm.sup.2) using a disc punch. Bismuth thiols were
resuspended in dimethyl sulfoxide (DMSO) and diluted to appropriate
concentrations for coating. Discs were coated with the bismuth
thiol solutions at a final concentration of 0.35 .mu.g/.mu.l using
50 .mu.l of the solution per disc for a final concentration of 10
.mu.g/cm.sup.2. The discs were subsequently placed in a lyophilizer
overnight to remove the DMSO solvent. S. aureus (ATCC #25983) was
used as the bacteria for this experiment. Bacteria was grown in
Tryptic Soy Broth for 16-24 hours before placing a lawn on Tryptic
Soy Agar in 100 mm Petri dishes.
Thirty six samples were tested in total (2 BT*n=3.fwdarw.6*6
rehydration times.fwdarw.36). A lawn of S. aureus was swabbed onto
the Tryptic Soy Agar. For the 0 rehydration time point, the bismuth
thiol coated disc was placed approximately in the center of the
plate and rehydrated with .about.50 .mu.l of sterile saline. Other
rehydration times were varied at 5, 10, 20, 30, or 60 minutes. For
this, the samples were placed in sterile saline and a timer set for
each time. At the appropriate time, samples were removed from the
saline and placed approximately in the center of the plate. Plates
were incubated inverted for 24 hours at which time pictures were
taken with a digital camera. Zones-of-inhibition were compared
visually between groups.
All of the BisEDT and BisBAL coated SIS discs illustrated
significant effectiveness in creating a zone-of-inhibition. There
were no visual differences between the rehydration time points.
This suggests that a variety of rehydration times can be utilized
on a medical device coated with a bismuth thiol without diminishing
the antimicrobial activity of the device.
EXAMPLE 4
This Example was performed to test the compatibility of BisEDT with
two sterilization techniques: ethylene oxide exposure or electron
beam (E-beam) irradiation.
BisEDT was obtained from Microbion, Inc. as previously described.
Small intestinal submucosa (SIS) was obtained and processed into
4-layer lyophilized sheets as described in Example 1. The sheets
were subsequently cut into 15 mm discs (.about.1.76 cm.sup.2) using
a disc punch. BisEDT was resuspended in dimethyl sulfoxide (DMSO)
and diluted to appropriate concentrations for coating. Discs were
coated with the BisEDT solution at a final concentration of 0.35
.mu.g/.mu.l using 50 .mu.l of the solution per disc for a final
concentration of 10 .mu.g/cm.sup.2. The discs were subsequently
placed in a lyophilizer overnight to remove the DMSO solvent. For
the ethylene oxide sterilization compatibility experiment, 6 discs
were coated with 10 .mu.g/cm.sup.2 BisEDT and packaged into 2
separate tyvek pouches with 3 discs per pouch. One pouch was placed
in a drawer until use and left non-sterile and the other pouch was
sterilized with low temperature ethylene oxide sterilization. For
the E-beam sterilization, essentially the same process was
performed except the discs were packaged in foil pouches and the
samples were subjected to E-beam sterilization with a dose of
.about.25 kGy. S. aureus (ATCC #25983) was used as the bacteria for
this experiment. Bacteria was grown in Tryptic Soy Broth for 16-24
hours before placing a lawn on Tryptic Soy Agar in 100 mm Petri
dishes.
The experiment used a total of 6 samples (3 sterilized, 3
non-sterile) for each sterilization protocol. A lawn of S. aureus
was swabbed onto the Tryptic Soy Agar. Each BisEDT coated disc was
rehydrated in sterile saline for .about.5 minutes then placed
approximately in the center of the plate. Plates were incubated
inverted for 24 hours at which time pictures were taken with a
digital camera. Zones-of-inhibition were compared visually between
groups.
All of the BisEDT coated SIS discs illustrated effectiveness at
creating a zone-of-inhibition. E-beam sterilization exhibited
greater compatibility with BisEDT than ethylene oxide
sterilization.
EXAMPLE 5
This Example was performed to test the ability of BisEDT to provide
anti-fungal activity.
BisEDT was obtained from Microbion, Inc. as previously described.
Small intestinal submucosa (SIS) was obtained and processed into
4-layer lyophilized sheets as described in Example 1. The sheets
were subsequently cut into 15 mm discs (.about.1.76 cm.sup.2) using
a disc punch. BisEDT was resuspended in dimethyl sulfoxide (DMSO)
and diluted to appropriate concentrations for coating. Discs were
coated with the BisEDT solution at a final concentration of 3.5
.mu.g/.mu.l using 50 .mu.l of the solution per disc for a final
concentration of 100 .mu.g/cm.sup.2. The discs were subsequently
placed in a lyophilizer overnight to remove the DMSO solvent. A
total of 6 coated discs were made--3 each for the two
microorganisms investigated. These were compared against 3 uncoated
controls discs cut from the same lot of SIS for each microorganism.
C. albicans (ATCC #10231) and A. niger(ATCC #16404) were plated on
Rose Bengal Chloramphenicol (RBC) in Petri dishes.
The experiment consisted of a total of 3 samples (3 BisEDT 100
.mu.g/cm.sup.2 coated, 3 uncoated controls) for each fungus. A lawn
of either C. albicans or A. niger was swabbed onto the RBC plates.
Each disc was rehydrated in sterile saline for .about.5 minutes
then placed approximately in the center of the plate. Plates were
incubated inverted for .about.48 hours for C. albicans or 5 and 8
days for A. niger at which time pictures were taken with a digital
camera. Zones-of-inhibition or growth inhibition was compared
visually between groups.
All of the BisEDT coated SIS discs illustrated some effectiveness
at growth inhibition. For C. albicans, the BisEDT coated discs had
a small zone-of-inhibition of .about.1 mm from the disc edge. The
uncoated controls had no zone-of-inhibition with the yeast growing
to the disc edge. This indicates that the BisEDT has some, albeit
minor, growth inhibiting capacity for this yeast. The mold, A.
niger, did not grow in a lawn but colonies were very evident. At 5
days, the mold was able to grow over the top of the control discs.
In contrast, the mold was able to grow up to the disc edge but not
on the BisEDT coated disc. These plates were incubated for an
additional 3 days to determine if this effect would last. Indeed,
at the eight day time point, the mold was still unable to grow on
the BisEDT coated discs.
This Example demonstrates that BisEDT confers some direct fungal
contact growth inhibition when delivered with an ECM. BisEDT
coating at 100 .mu.g/cm.sup.2 was able to inhibition growth of both
a yeast, C. albicans, and a mold, A. niger.
EXAMPLE 6
This Example was performed to determine the consistency of DMSO
absorption into 4-layer lyophilized SIS constructs.
Ten lots of SIS were (Cook Biotech Incorporated, West Lafayette,
Ind.) obtained and each was used to prepare a 7 cm.times.20 cm
four-layer lyophilized sheet as described in Example 1. Three 4
cm.times.4 cm pieces were cut from each sheet for a total of 30
samples. DMSO (Sigma Aldrich) was standard USP grade.
Each sample was weighed and weight recorded. Subsequently, each
sample was placed into an excess of DMSO and allowed to absorb the
fluid for 3 minutes. After 3 minutes, the sample was blotted to
remove excess DMSO and weighed, with the weight recorded as final
weight. DMSO content was calculated as final minus initial weight.
DMSO volume was calculated as DMSO weight divided density (1.1
g/ml). Percent DMSO was calculated as DMSO weight divided by the
total weight.
The processed 4-layer lyophilized SIS samples absorbed DMSO very
quickly. Each piece was allowed to absorb DMSO for three minutes to
give an excess of time to ensure full absorption. Initial sample
weights varied from 97.6 mg to 175 mg, with an average of 134 mg
and standard deviation of 19 mg. This indicates that the dry weight
of individual lots of SIS can vary significantly. The final amount
of DMSO absorbed varied from 416 mg to 768 mg with an average of
550 mg and standard deviation of 96 mg. This corresponds to a total
volume absorbed on average of 500 mg and standard deviation of 88
mg. This analysis illustrated a significant variation on amount of
DMSO absorbed for identical areas of SIS, indicating that excess
volume loading with only one concentration used will likely lead to
significant variation of substance absorbed onto the final device.
To determine if there was a better predictor, the DMSO percent of
total was calculated. This gave a result with less variation (80.2%
average with 3.1% standard deviation). Results are detailed in
Table 6.
TABLE-US-00006 TABLE 6 Initial DMSO DMSO Weight Final Weight weight
Volume Percent Lot # Sample (mg) (mg) (mg) (ml) DMSO P107828 a 98
517 420 382 81.1% b 111 608 496 451 81.7% c 109 544 435 396 80.0%
P107829 a 150 621 471 428 75.9% b 140 581 441 401 75.9% c 142 672
530 482 78.8% P107830 a 130 674 544 495 80.7% b 141 628 487 443
77.6% c 138 676 538 489 79.6% P107831 a 118 763 645 586 84.6% b 114
773 658 599 85.2% c 124 698 574 522 82.2% P107832 a 170 797 628 571
78.7% b 147 703 556 505 79.1% c 139 670 532 483 79.3% P107833 a 142
649 507 461 78.1% b 131 774 643 584 83.1% c 130 758 628 571 82.8%
P107834 a 165 740 574 522 77.6% b 122 537 415 377 77.2% c 139 647
509 462 78.6% P107835 a 117 885 768 698 86.7% b 119 921 802 729
87.1% c 119 751 632 574 84.1% P107836 a 129 590 461 419 78.2% b 120
591 472 429 79.8% c 118 555 437 397 78.7% P107837 a 175 780 605 550
77.6% b 167 742 574 522 77.4% c 156 686 530 482 77.3% Average 134
684 550 500 80.2% St Dev 19 100 96 88 3.1%
This Example demonstrates that DMSO is absorbed into SIS very
quickly. The amount of DMSO absorbed varied for the ten lots
tested. This suggests that if an excess volume of solvent is used,
the ending substance absorbed will vary significantly. Thus, care
must be taken if it is to be ensured that consistent bismuth thiol
loading onto a medical device is achieved when DMSO is used as a
solvent.
EXAMPLE 7
The Example was performed to compare the in vivo angiogenic
response to SIS compared to 2 levels of BisEDT coated SIS. The
model used to detect in vivo angiogenesis was the mouse
subcutaneous implant method as generally described by Heeschen et
al., Nat. Med. 200; 7:833-9.
BisEDT was obtained from Microbion, Inc. as previously described.
All SIS based grafts were made from 4-layer lyophilized sheets as
described in the previous Examples. Sample groups consisted of this
material coated with 10 .mu.g/cm.sup.2 BisEDT or 100 .mu.g/cm.sup.2
BisEDT. Each material was cut into ten 15 mm discs. Nylon filters
with 0.22 .mu.m pores were sewn on to the top and bottom of each
disc. Low temperature ethylene oxide sterilization was used for
each sample.
Samples were implanted subcutaneously into the dorsal flanks of
mice. After anesthesia using Ketamine (87 mg/kg) and Xylazine (13
mg/kg), a small incision was made on the posterior neck of the
mouse and a dorsal subcutaneous cavity was created using blunt
dissection with hemostats. This was followed by sample placement
and closure of the incision with 4 interrupted stitches of 5-0
suture. Six mice per group underwent disc implantation. The implant
remained in the mice for a period of 3 weeks followed by probing
for capillary formation.
Mice were sacrificed using a double dose of anesthesia to ensure
intact flow in vasculature. While the heart was still beating, the
chest cavity was exposed, vena cava severed, and 10 ml of heparized
saline injected into the left ventricle using a 23 ga butterfly
infusion set to exsanguinate the mouse. After transferring syringes
(while maintaining infusion needle in left ventricle), 4 ml of a
fluorescent microsphere (yellow-green, 0.1 .mu.m diameter,
Molecular Probes, F-8803) suspension (1:20 dilution of stock
suspension) was injected through the left ventricle resulting in
perfusion of the entire vasculature. Care was taken to ensure no
bubbles were introduced during the injections, as bubbles will
cause micro-emboli obstructing consistent perfusion. Samples were
collected with gentle dissection and gross removal of the fibrous
capsule. A positive control of hind limb muscle was also collected
at this point to confirm proper perfusion. Collected samples and
controls were placed on ice in a closed container to maintain
tissue integrity (mainly moistness). Microvasculature was imaged
using a confocal microscope (Biorad), .lamda..sub.ex=488 nm &
.lamda..sub.em=530 nm, along the edge of the samples in the area of
greatest vascular infiltration. Further, vasculature of the
positive controls, hind limb muscle, was imaged to confirm good
perfusion.
In addition to the above, samples were collected, placed in
histology cassettes, and submerged in 10% buffered formalin
(Fisher). Histological sectioning and staining with hematoxilin and
eosin were performed by Portland Tissue Processing. Images of
H&E stained sections of the disc edge for each sample were
taken using a microscrope (Olympus) with a 10.times. objective.
Lyophilized control SIS performed with robust angiogenesis evident
burrowing multiple millimeters into the disc. Further, it was
discovered that neither level of BisEDT (10 or 100 .mu.g/cm.sup.2)
coated onto the SIS affected the angiogenic potential. In addition,
there were no observed symptoms of systemic toxicity, and no visual
evidence of local toxicity at explant for these samples, indicating
that these levels of BisEDT are well tolerated.
This Example demonstrates that both BisEDT concentration samples
had similar angiogenesis and tissue ingrowth compared to control
without signs of systemic or local toxicity or local inflammation.
BisEDT is well tolerated and do not negatively affect remodeling of
the ECM.
It is intended that the foregoing detailed description be regarded
as illustrative rather than limiting, and that it be understood
that it is the following claims, including all equivalents, that
are intended to define the spirit and scope of this invention.
* * * * *